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7884d_c15.qxd 1/23/03 Chapter 15 12:27 PM Page 496 mac18 mac18:df_169:7884D: Enzymatic Catalysis 1 Catalytic Mechanisms A. Acid–Base Catalysis B. Covalent Catalysis C. Metal Ion Catalysis D. Electrostatic Catalysis E. Catalysis through Proximity and Orientation Effects F. Catalysis by Preferential Transition State Binding 2 Lysozyme A. Enzyme Structure B. Catalytic Mechanism C. Testing the Phillips Mechanism 3 Serine Proteases A. Kinetics and Catalytic Groups B. X-Ray Structures C. Catalytic Mechanism D. Testing the Catalytic Mechanism E. Zymogens 4 Drug Design A. Techniques of Drug Discovery B. Introduction to Pharmacology C. HIV Protease and Its Inhibitors Enzymes, as we have seen, cause rate enhancements that are orders of magnitude greater than those of the best chemical catalysts. Yet they operate under mild conditions and are highly specific as to the identities of both their substrates and their products. These catalytic properties are so remarkable that many nineteenth century scientists concluded that enzymes have characteristics that are not shared by substances of nonliving origin. To this day, there are few enzymes for which we understand in more than cursory detail how they achieve their enormous rate accelerations. Nevertheless, it is now abundantly clear that the catalytic mechanisms employed by enzymes are identical to those used by chemical catalysts. Enzymes are simply better designed. In this chapter we consider the nature of enzymatic catalysis. We begin by discussing the underlying principles of chemical catalysis as elucidated through the study of organic reaction mechanisms. We then embark on a detailed examination of the catalytic mechanisms of several of the best characterized enzymes: lysozyme and the serine proteases. Their study should lead to an appreciation of the intracacies of these remarkably efficient catalysts as well as of the experimental methods used to elucidate their 496 properties. We end with a discussion of how drugs are discovered and tested, a process that depends heavily on the principles of enzymology since many drug targets are enzymes. In doing so, we consider how therapeutically effective inhibitors of HIV-1 protease were discovered. 1 CATALYTIC MECHANISMS Catalysis is a process that increases the rate at which a reaction approaches equilibrium. Since, as we discussed in Section 14-1C, the rate of a reaction is a function of its free energy of activation (G ‡), a catalyst acts by lowering the height of this kinetic barrier; that is, a catalyst stabilizes the transition state with respect to the uncatalyzed reaction. There is, in most cases, nothing unique about enzymatic mechanisms of catalysis in comparison to nonenzymatic mechanisms. What apparently make enzymes such powerful catalysts are two related properties: their specificity of substrate binding combined with their optimal arrangement of catalytic groups. An enzyme’s arrangement of binding and catalytic groups is, of course, the product of eons of evolution: Nature has had ample opportunity to finetune the performances of most enzymes. The types of catalytic mechanisms that enzymes employ have been classified as: 1. Acid–base catalysis. 2. Covalent catalysis. 3. Metal ion catalysis. 4. Electrostatic catalysis. 5. Proximity and orientation effects. 6. Preferential binding of the transition state complex. In this section, we examine these various phenomena. In doing so we shall frequently refer to the organic model compounds that have been used to characterize these catalytic mechanisms. A. Acid–Base Catalysis General acid catalysis is a process in which partial proton transfer from a Brønsted acid (a species that can donate protons; Section 2-2A) lowers the free energy of a reaction’s transition state. For example, an uncatalyzed keto–enol tautomerization reaction occurs quite slowly as a result of 1/29/03 12:01 PM Page 497 mac46 mac46:1352_mjw:7884d: Section 15–1. Catalytic Mechanisms Keto Transition state R (a) C O CH2 δ– CH 2 H H C O + H O H δ+ A δ– C δ– H H H2O B A + OH – R δ– O H .. B – A H δ– CH 2 + + δ+ C H H CH2 H+ R CH2 O R CH 2 O H CH2 δ– C A O δ+ CH2 C C R R (c) R δ– C R (b) Enol R O 497 C + H O H CH2 + δ+ + H H δ+ B + .. 7884d_c15.qxd + B FIGURE 15-1 Mechanisms of keto–enol tautomerization. (a) Uncatalyzed, (b) general acid catalyzed, and (c) general base catalyzed. the high energy of its carbanionlike transition state (Fig. 15-1a). Proton donation to the oxygen atom (Fig. 15-1b), however, reduces the carbanion character of the transition state, thereby catalyzing the reaction. A reaction may also be stimulated by general base catalysis if its rate is increased by partial proton abstraction by a Bro/nsted base (a species that can combine with a proton; Fig. 15-1c). Some reactions may be simultaneously subject to both processes: a concerted general acid–base catalyzed reaction. a. Mutarotation Is Catalyzed by Acids and by Bases The mutarotation of glucose provides an instructive example of acid–base catalysis. Recall that a glucose molecule can assume either of two anomeric cyclic forms through the intermediacy of its linear form (Section 11-1B): H CH2OH O H OH H H H OH HO CH2OH O H OH H H OH -D-Glucose []20 D = 18.7 H CH2OH OH H CH OH H HO v d3-D-glucose 4 dt H O H A O H A O H C O H H B– -D-Glucose -D-Glucose H A– O [15.1] where kobs is the reaction’s apparent first-order rate constant. The mutarotation rate increases with the concentrations of general acids and general bases; they are thought to catalyze mutarotation according to the mechanism: O CH H OH Linear form kobs 3-D-glucose4 C H HO H OH -D-Glucose []20 D = 112.2 OH In aqueous solvents, the initial rate of mutarotation of -D-glucose, as monitored by polarimetry (Section 4-2A), is observed to follow the relationship: O H Linear form B B– 7884d_c15.qxd 498 1/29/03 12:01 PM Page 498 mac46 mac46:1352_mjw:7884d: Chapter 15. Enzymatic Catalysis This model is consistent with the observation that in aprotic solvents such as benzene, 2,3,4,6-O-tetramethyl-D-glucose (a less polar benzene-soluble analog) H H3CO CH2OCH3 O H H OCH3 H OH drolyze RNA to its component nucleotides. The isolation of 2,3-cyclic nucleotides from RNase A digests of RNA indicates that the enzyme mediates the following reaction sequence: O –O P H OCH3 2,3,4,6-O-Tetramethyl--D-glucose 5 O CH2 4 O O H Base OH O –O RNA H 2 3 does not undergo mutarotation. Yet, the reaction is catalyzed by the addition of phenol, a weak benzene-soluble acid, together with pyridine, a weak benzene-soluble base, according to the rate equation: 1 H H P O CH2 O v k3 phenol 4 3pyridine 4 3tetramethyl--D-glucose4 [15.2] O Base H H O OH H Moreover, in the presence of -pyridone, whose acid and base groups can rapidly interconvert between two tautomeric forms and are situated so that they can simultaneously catalyze mutarotation, –O H P O O -Pyridone O N N O H H O H –O P O CH2 O H O Base H H H O O C O H O O C H O + P –O H HO CH2 O H 2,3-Cyclic nucleotide H –O v k¿ 3-pyridone 4 3tetramethyl--D-glucose4 [15.3] H O the reaction follows the rate law b. The RNase A Reaction Incorporates General Acid–Base Catalysis Bovine pancreatic ribonuclease A (RNase A) provides an illuminating example of enzymatically mediated general acid–base catalysis. This digestive enzyme functions to hy- Base H Glucose where k 7000M k. This increased rate constant indicates that -pyridone does, in fact, catalyze mutarotation in a concerted fashion since 1M -pyridone has the same catalytic effect as impossibly high concentrations of phenol and pyridine (e.g., 70M phenol and 100M pyridine). Many types of biochemically significant reactions are susceptible to acid and/or base catalysis. These include the hydrolysis of peptides and esters, the reactions of phosphate groups, tautomerizations, and additions to carbonyl groups. The side chains of the amino acid residues Asp, Glu, His, Cys, Tyr, and Lys have pK’s in or near the physiological pH range (Table 4-1) which, we shall see, permits them to act in the enzymatic capacity of general acid and/or base catalysts in analogy with known organic mechanisms. Indeed, the ability of enzymes to arrange several catalytic groups about their substrates makes concerted acid–base catalysis a common enzymatic mechanism. O H2O H+ P OH O O O –O P O CH2 O H O Base H H H O –O P OH O O– The RNase A reaction exhibits a pH rate profile that peaks near pH 6 (Fig. 15-2). Analysis of this curve (Section 14-4), together with chemical derivatization and X-ray studies, indicates that RNase A has two essential His residues, His 12 and His 119, which act in a concerted manner as general acid and base catalysts (the structure of RNase A is sketched in Fig. 9-2). Evidently, the RNase A reaction is a two-step process (Fig. 15-3): 1. His 12, acting as a general base, abstracts a proton from an RNA 2-OH group, thereby promoting its nucleo- 1/23/03 12:27 PM Page 499 mac18 mac18:df_169:7884D: Section 15–1. Catalytic Mechanisms FIGURE 15-2 The pH dependence of V¿max K¿M in the RNase A–catalyzed hydrolysis of cytidine-2,3-cyclic phosphate. V¿max K¿M is given in units of M1 s1. Analysis of this curve (Section 14-4) suggests the catalytic participation of groups with pK’s of 5.4 and 6.4. [After del Rosario, E.J. and Hammes, G.G., Biochemistry 8, 1887 (1969).] 499 5 log V'max K'M 3 pKE1 pKE2 1 4 5 6 7 8 ... pH ... O P –O O CH2 O 4 O H Base H O –O P H O O H His 119 N H –O H P O N+ NH P H OH O H –O O O H O H O 1 HO N + H Base H Base H H CH2 O H2O His 12 CH2 O O NH N 2 3 O O 1 H H P CH2 O H H Base S –O 2′,3′-Cyclic nucleotide O RNA 5 H H O –O N H H O ... P N H OH O O ... 2 ... 7884d_c15.qxd O –O P O CH2 O H O Base H H H O –O P O O H H N+ The bovine pancreatic RNase A–catalyzed hydrolysis of RNA is a two-step process with the intermediate formation of a 2,3-cyclic nucleotide. FIGURE 15-3 O N H H SN NH 9 7884d_c15.qxd 1/29/03 12:01 PM Page 500 mac46 mac46:1352_mjw:7884d: Chapter 15. Enzymatic Catalysis 500 philic attack on the adjacent phosphorus atom while His 119, acting as a general acid, promotes bond scission by protonating the leaving group. 2. The 2,3-cyclic intermediate is hydrolyzed through what is essentially the reverse of the first step in which water replaces the leaving group. Thus His 12 acts as a general acid and His 119 as a general base to yield the hydrolyzed RNA and the enzyme in its original state. B. Covalent Catalysis Covalent catalysis involves rate acceleration through the transient formation of a catalyst–substrate covalent bond. The decarboxylation of acetoacetate, as chemically catalyzed by primary amines, is an example of such a process (Fig. 15-4). In the first stage of this reaction, the amine nucleophilically attacks the carbonyl group of acetoacetate to form a Schiff base (imine bond). H H + N C H + N C O N C OH H H B + OH – Schiff base A The protonated nitrogen atom of the covalent intermediate then acts as an electron sink (Fig. 15-4, bottom) so as to reduce the otherwise high-energy enolate character of the transition state. The formation and decomposition of the Schiff base occur quite rapidly, so that these steps are not rate determining in this reaction sequence. a. Covalent Catalysis Has Both Nucelophilic and Electrophilic Stages As the preceding example indicates, covalent catalysis may be conceptually decomposed into three stages: CO2 O CH3 C CH2 C CH3 C 2. The withdrawal of electrons from the reaction center by the now electrophilic catalyst. 3. The elimination of the catalyst, a reaction that is essentially the reverse of stage 1. Reaction mechanisms are somewhat arbitrarily classified as occurring with either nucleophilic catalysis or electrophilic catalysis depending on which of these effects provides the greater driving force for the reaction, that is, which catalyzes its rate-determining step. The primary amine–catalyzed decarboxylation of acetoacetate is clearly an electrophilically catalyzed reaction since its nucleophilic phase, Schiff base formation, is not its rate-determining step. In other covalently catalyzed reactions, however, the nucleophilic phase may be rate determining. The nucleophilicity of a substance is closely related to its basicity. Indeed, the mechanism of nucleophilic catalysis resembles that of general base catalysis except that, instead of abstracting a proton from the substrate, the catalyst nucleophilically attacks it so as to form a covalent bond. Consequently, if covalent bond formation is the ratedetermining step of a covalently catalyzed reaction, the reaction rate tends to increase with the covalent catalyst’s basicity (pK). An important aspect of covalent catalysis is that the more stable the covalent bond formed, the less facilely it will decompose in the final steps of a reaction. A good covalent catalyst must therefore combine the seemingly contradictory properties of high nucleophilicity and the ability to form a good leaving group, that is, to easily reverse the bond formation step. Groups with high polarizabilities (highly mobile electrons), such as imidazole and thiol functions, have these properties and hence make good covalent catalysts. + H –O O 1. The nucleophilic reaction between the catalyst and the substrate to form a covalent bond. CH2 O CH3 C CH3 – O Acetoacetate OH Acetone Enolate RNH2 RNH2 OH CH3 N H C CH2 CO2 R CH3 – O Schiff base (imine) H N O C .. R C CH2 H+ R CH3 N H C CH3 FIGURE 15-4 The decarboxylation of acetoacetate. The uncatalyzed reaction mechanism is shown at the top and the reaction mechanism as catalyzed by primary amines is shown at the bottom. 7884d_c15.qxd 1/23/03 12:27 PM Page 501 mac18 mac18:df_169:7884D: Section 15–1. Catalytic Mechanisms b. Certain Amino Acid Side Chains and Coenzymes Can Serve as Covalent Catalysts Enzymes commonly employ covalent catalytic mechanisms as is indicated by the large variety of covalently linked enzyme–substrate reaction intermediates that have been isolated. For example, the enzymatic decarboxylation of acetoacetate proceeds, much as described above, through Schiff base formation with an enzyme Lys residue’s e-amino group. The covalent intermediate, in this case, has been isolated through NaBH4 reduction of its imine bond to an amine, thereby irreversibly inhibiting the enzyme. Other enzyme functional groups that participate in covalent catalysis include the imidazole moiety of His, the thiol group of Cys, the carboxyl function of Asp, and the hydroxyl group of Ser. In addition, several coenzymes, most notably thiamine pyrophosphate (Section 17-3B) and pyridoxal phosphate (Section 26-1A), function in association with their apoenzymes mainly as covalent catalysts. 501 The decarboxylation of dimethyloxaloacetate, as catalyzed by metal ions such as Cu2 and Ni2, is a nonenzymatic example of catalysis by a metal ion: M n+ –O O O CH3 C C C O C O– CH3 Dimethyloxaloacetate CO2 M n+ O– CH3 –O C C C O CH3 C. Metal Ion Catalysis Nearly one-third of all known enzymes require the presence of metal ions for catalytic activity. There are two classes of metal ion–requiring enzymes that are distinguished by the strengths of their ion–protein interactions: H+ –O O C 1. Metalloenzymes contain tightly bound metal ions, most commonly transition metal ions such as Fe2, Fe3, Cu2, Zn2, Mn2, or Co3. 2. Metal-activated enzymes loosely bind metal ions from solution, usually the alkali and alkaline earth metal ions Na, K, Mg2, or Ca2. Metal ions participate in the catalytic process in three major ways: 1. By binding to substrates so as to orient them properly for reaction. 2. By mediating oxidation–reduction reactions through reversible changes in the metal ion’s oxidation state. 3. By electrostatically stabilizing or shielding negative charges. In this section we shall be mainly concerned with the third aspect of metal ion catalysis. The other forms of enzyme-mediated metal ion catalysis are considered in later chapters in conjunction with discussions of specific enzyme mechanisms. a. Metal Ions Promote Catalysis through Charge Stabilization In many metal ion–catalyzed reactions, the metal ion acts in much the same way as a proton to neutralize negative charge, that is, it acts as a Lewis acid. Yet metal ions are often much more effective catalysts than protons because metal ions can be present in high concentrations at neutral pH’s and can have charges greater than 1. Metal ions have therefore been dubbed “superacids.” O C CH3 CH + Mn+ CH3 Here the metal ion (Mn), which is chelated by the dimethyloxaloacetate, electrostatically stabilizes the developing enolate ion of the transition state. This mechanism is supported by the observation that acetoacetate, which cannot form such a chelate, is not subject to metal ion– catalyzed decarboxylation. Most enzymes that decarboxylate oxaloacetate require a metal ion for activity. b. Metal Ions Promote Nucleophilic Catalysis via Water Ionization A metal ion’s charge makes its bound water molecules more acidic than free H2O and therefore a source of OH ions even below neutral pH’s. For example, the water molecule of (NH3)5Co3(H2O) ionizes according to the reaction: 1NH3 2 5Co3 1H2O2 ∆ 1NH3 2 5Co3 1OH 2 H with a pK of 6.6, which is 9 pH units below the pK of free H2O. The resulting metal ion–bound hydroxyl group is a potent nucleophile. An instructive example of this phenomenon occurs in the catalytic mechanism of carbonic anhydrase (Section 10-1C), a widely occurring enzyme that catalyzes the reaction: CO2 H 2O ∆ HCO3 H Carbonic anhydrase contains an essential Zn2 ion that lies at the bottom of an 15-Å-deep active site cleft (Fig. 8-41), where it is tetrahedrally coordinated by three evolutionarily invariant His side chains and an O atom of either an 7884d_c15.qxd 3/24/03 1:01 PM Page 502 mac85 Mac 85:1st shift: 1268_tm:7884d: 502 Chapter 15. Enzymatic Catalysis HCO3 ion (Fig. 15-5a) or a water molecule (Fig. 15-5b). The enzyme has the following catalytic mechanism: 1. We begin with a water molecule bound to the protein in the Zn2 ion’s fourth liganding position (Fig. 15-5b). This Zn2-polarized H2O ionizes in a process facilitated through general base catalysis by His 64 in its “in” conformation. Although His 64 is too far away from the Zn2-bound water to directly abstract its proton, these entities are linked by two intervening water molecules to form a hydrogen bonded network that is thought to act as a proton shuttle. H H H N N H O H O H H O Zn2+ Im Im Im (a) His 64 H H N H N + O H H H H O– Im Zn2+ O Im Im His 64 Im = imidazole 2. The resulting Zn2-bound OH ion nucleophilically attacks the nearby enzymatically bound CO2, thereby converting it to HCO3 . Im Im Zn (b) 2+ Im FIGURE 15-5 X-Ray structures of human carbonic anhydrase. (a) Its active site in complex with bicarbonate ion. The polypeptide is shown in ribbon form (gold) with its side chains shown in stick form colored according to atom type (C green, N blue, and O red). The protein-bound Zn2 ion (cyan sphere) is tetrahedally liganded (gray bonds) by three invariant His side chains and the HCO3 ion, which is shown in ball-and-stick form. The HCO3 ion also interacts with the protein via van der Waals contacts (dot surface colored according to atom type) and a hydrogen bonded network (dashed gray lines) involving Thr 199 and Glu 106. [Based on an X-ray structure by K. K. Kannan, Bhabha Atomic Research Center, Bombay, India. PDBid 1HCB.] (b) The active site showing the proton shuttle through which His 64, acting as a general base, abstracts a proton from the Zn2-bound H2O to form an OH ion. The polypeptide backbone is shown in ribbon form (cyan), and its side chains and several bound solvent molecules are shown in ball-and-stick form with C black, N blue, and O red. The proton shuttle consists of two water molecules that form a hydrogen bonded network (dotted white lines) that bridges the Zn2-bound OH ion and His 64 in its “in” conformation. On protonation, His 64 swings to the “out” conformation. [Courtesy of David Christianson, University of Pennsylvania.] See the Interactive Exercises O O– + C O H Im Im O Zn2+ O Im H C O– H2O Im Im Zn 2+ Im O O– H + H+ + H O C O– Im = imidazole In doing so, the Zn2-bound OH group donates a hydrogen bond to Thr 199, which in turn donates a hydrogen bond to Glu 106 (Fig. 15-5a). These interactions orient the OH group with the optimal geometry (see below) for nucleophilic attack on the substrate CO2. 3. The catalytic site is regenerated by the exchange of the Zn2-bound HCO3 reaction product for H2O together 7884d_c15.qxd 1/23/03 12:27 PM Page 503 mac18 mac18:df_169:7884D: Section 15–1. Catalytic Mechanisms with the deprotonation of His 64. In the latter process, His 64 swings to its “out” conformation (Fig. 15-5b), which may facilitate proton transfer to the bulk solvent. 503 the bimolecular reaction of imidazole with p-nitrophenylacetate, O c. Metal Ions Promote Reactions through Charge Shielding Another important enzymatic function of metal ions is charge shielding. For example, the actual substrates of kinases (phosphoryl-transfer enzymes utilizing ATP) are Mg2 –ATP complexes such as C CH3 Adenine Ribose O P O O– O P O P (p-NO2Ac) NH O– O NO2 p-Nitrophenylacetate N O Mg2+ O– O Imidazole O– k1 O rather than just ATP. Here, the Mg2 ion’s role, in addition to its orienting effect, is to shield electrostatically the negative charges of the phosphate groups. Otherwise, these charges would tend to repel the electron pairs of attacking nucleophiles, especially those with anionic character. O + C CH3 _ O NO2 N + p-Nitrophenolate (p-NO2O) NH N-Acetylimidazolium D. Electrostatic Catalysis The binding of substrate generally excludes water from an enzyme’s active site. The local dielectric constant of the active site therefore resembles that in an organic solvent, where electrostatic interactions are much stronger than they are in aqueous solutions (Section 8-4A). The charge distribution in a medium of low dielectric constant can greatly influence chemical reactivity. Thus, as we have seen, the pK’s of amino acid side chains in proteins may vary by several units from their nominal values (Table 4-1) because of the proximity of charged groups. Although experimental evidence and theoretical analyses on the subject are still sparse, there are mounting indications that the charge distributions about the active sites of enzymes are arranged so as to stabilize the transition states of the catalyzed reactions. Such a mode of rate enhancement, which resembles the form of metal ion catalysis discussed above, is termed electrostatic catalysis. Moreover, in several enzymes, these charge distributions apparently serve to guide polar substrates toward their binding sites so that the rates of these enzymatic reactions are greater than their apparent diffusion-controlled limits (Section 14-2B). E. Catalysis through Proximity and Orientation Effects Although enzymes employ catalytic mechanisms that resemble those of organic model reactions, they are far more catalytically efficient than these models. Such efficiency must arise from the specific physical conditions at enzyme catalytic sites that promote the corresponding chemical reactions. The most obvious effects are proximity and orientation: Reactants must come together with the proper spatial relationship for a reaction to occur. For example, in the progress of the reaction is conveniently monitored by the appearance of the intensely yellow p-nitrophenolate ion: d3p-NO2 O 4 dt k1 3 imidazole4 3p-NO2 Ac4 [15.4] k¿1 3p-NO2 Ac4 where phenyl. Here k¿1, the pseudo-first-order rate constant, is 0.0018 s1 when [imidazole] 1M. However, for the intramolecular reaction O C O O NO2 N NH k2 C + N+ –O NO2 NH the first-order rate constant k2 0.043 s1; that is, k2 24k¿1. Thus, when the 1M imidazole catalyst is covalently attached to the reactant, it is 24-fold more effective than when it is free in solution; that is, the imidazole group in the intramolecular reaction behaves as if its concentration is 24M. This rate enhancement has contributions from both proximity and orientation. a. Proximity Alone Contributes Relatively Little to Catalysis Let us make a rough calculation as to how the rate of a reaction is affected purely by the proximity of its reacting groups. Following Daniel Koshland’s treatment, we shall make several reasonable assumptions: 1. Reactant species, that is, functional groups, are about the size of water molecules. 7884d_c15.qxd 504 2/1/03 4:50 PM Page 504 mac 106 mac 106:211_sks: Chapter 15. Enzymatic Catalysis Thus, in the absence of other effects, this model predicts that for the intramolecular reaction, 2. Each reactant species in solution has 12 nearestneighbor molecules, as do packed spheres of identical size. 3. Chemical reactions occur only between reactants that are in contact. A 4. The reactant concentration in solution is low enough so that the probability of any reactant species being in simultaneous contact with more than one other reactant molecule is negligible. k1 A B ¡ A¬ B dt k1 3A4 3B 4 k2 3A, B4 pairs [15.5] where [A,B]pairs is the concentration of contacting molecules of A and B. The value of this quantity is 3A, B4 pairs 12 3A4 3B 4 [15.6] 55.5M since there are 12 ways that A can be in contact with B, and [A]55.5M is the fraction of sites occupied by A in water solution ([H2O] 55.5M in dilute aqueous solutions) and hence the probability that a molecule of B will be next to one of A. Combining Eqs. [15.5] and [15.6] yields v k1a 55.5 b3 A, B4 pairs 4.6k1 3A, B4 pairs 12 [15.7] R C R δ– X X B b. Properly Orienting Reactants and Arresting Their Relative Motions Can Result in Large Catalytic Rate Enhancements The foregoing theory is, of course, quite simple. For example, it does not take into account the relative orientations of the reacting molecules. Yet molecules are not equally reactive in all directions as Koshland’s simple theory assumes. Rather, they react most readily only if they have the proper relative orientation. For example, in an SN2 (bimolecular nucleophilic substitution) reaction, the incoming nucleophile optimally attacks its target C atom along the direction opposite to that of the bond to the leaving group (Fig. 15-6). The approaches of reacting atoms along a trajectory that deviates by as little as 10 from this optimum direction can reduce the reaction rate by as much as a factor of 100. In a related phenomenon, a molecule may be maximally reactive only when it assumes a conformation that aligns its various orbitals in a way that minimizes the electronic energy of its transition state, an effect termed stereoelectronic assistance. obeys the second-order rate equation d3A¬ B4 A k2 4.6k1, which is a rather small rate enhancement. Factors that will increase this value other than proximity alone clearly must be considered. Then the reaction: v k2 B X– R′′ C R′′ R C R′ R′ R′′ R′ Yδ – Y– Y sp2–p hybridization at carbon The geometry of an SN2 reaction. The attacking nucleophile, Y, must approach the tetrahedrally coordinated and hence sp3-hybridized C atom along the direction opposite that of its bond to the leaving group, X, a process called backside attack. In the transition state of the reaction, the C atom becomes trigonal bipyramidally coordinated and hence sp2 –p hybridized, with the p orbital (blue) forming partial bonds to X and Y. The three sp2 orbitals form bonds to the C atom’s FIGURE 15-6 three other substituents (R, R¿, and R– ), which have shifted their positions into the plane perpendicular to the X¬ C¬ Y axis (curved arrows). Any deviation from this optimal geometry would increase the free energy of the transition state, G‡, and hence reduce the rate of the reaction (Eq. [14.15]). The transition state then decomposes to products in which the R, R, and R have inverted their positions about the C atom, which has rehybridized to sp3, and X has been released. 1/23/03 12:27 PM Page 505 mac18 mac18:df_169:7884D: Section 15–1. Catalytic Mechanisms Another effect that we have neglected in our treatment of proximity is that of motions of the reacting groups with respect to one another. Yet, in the transition state complex, the reacting groups have little relative motion. In fact, as Thomas Bruice demonstrated, the rates of intramolecular reactions are greatly increased by arresting a molecule’s internal motions in a way that increases the mole fraction of the reacting groups that are in a conformation which can enter the transition state (Table 15-1). Similarly, when an enzyme brings two molecules together in a bimolecular reaction, as William Jencks pointed out, not only does it increase their proximity, but it freezes out their relative translational and rotational motions (decreases their entropy), thereby enhancing their reactivity. Theoretical studies by Bruice indicate that much of this rate enhancement can arise from the enzymatic binding of substrates in a conformation that readily enters the transition state. Enzymes, as we shall see in Sections 16-2 and 16-3, bind substrates in a manner that both aligns and immobilizes them so as to optimize their reactivities. The free energy required to do so is derived from the specific binding free energy of substrate to enzyme. O R1 C O + R2 C Br O O R1 C O _ R2 + _O O O Reactantsa Relative Rate Constant CH3COO Br + 1.0 _ CH3COO COO Br 1 103 _ COO COO Br 2.3 105 _ COO O R H a C COOH C O R Steric strain R Curved arrows indicate rotational degrees of freedom. Source: Bruice, T.C. and Lightstone, F.C., Acc. Chem. Res. 32, 127 (1999). as cyclopropane than for unstrained rings such as cyclohexane. In either process, the strained reactant more closely resembles the transition state of the reaction than does the corresponding unstrained reactant. Thus, as was first suggested by Linus Pauling and further amplified by Richard Wolfenden and Gustav Lienhard, interactions that preferentially bind the transition state increase its concentration and therefore proportionally increase the reaction rate. Let us quantitate this statement by considering the kinetic consequences of preferentially binding the transition state of an enzymatically catalyzed reaction involving a single substrate. The substrate S may react to form product P either spontaneously or through enzymatic catalysis: kN H CH2OH 8 107 _ COO The rate enhancements effected by enzymes are often greater than can be reasonably accounted for by the catalytic mechanisms so far discussed. However, we have not yet considered one of the most important mechanisms of enzymatic catalysis: the binding of the transition state to an enzyme with greater affinity than the corresponding substrates or products. When taken together with the previously described catalytic mechanisms, preferential transition state binding rationalizes the observed rates of enzymatic reactions. The original concept of transition state binding proposed that enzymes mechanically strained their substrates toward the transition state geometry through binding sites into which undistorted substrates did not properly fit. This so-called rack mechanism (in analogy with the medieval torture device) was based on the extensive evidence for the role of strain in promoting organic reactions. For example, the rate of the reaction, R Br C COO Br F. Catalysis by Preferential Transition State Binding 505 TABLE 15-1 Relative Rates of Anhydride Formation for Esters Possessing Different Degrees of Motional Freedom in the Reaction: S ¡ P kE ES ¡ EP + H2O O Here kE and kN are the first-order rate constants for the catalyzed and uncatalyzed reactions, respectively. The relationships between the various states of these two reaction pathways are indicated in the following scheme: ‡ KN KR ‡ KE KT ES ∆ ES‡ ¡ ∆ is 315 times faster when R is CH3 rather than when it is H because of the greater steric repulsions between the CH3 groups and the reacting groups. Similarly, ring opening reactions are considerably more facile for strained rings such ∆ ‡ ES ∆ S E ¡ PE ∆ 7884d_c15.qxd EP 7884d_c15.qxd 506 1/23/03 12:28 PM Page 506 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis where E+S KR ‡ KN 3ES 4 3E4 3S4 3E4 3S‡ 4 3E4 3S 4 KT and ‡ KE 3ES‡ 4 3 E4 3S‡ 4 3ES‡ 4 ∆GN 3 ES4 ES G are all association constants. Consequently, ‡ 3S4 3ES‡ 4 KE KT ‡ ‡ KR 3S 4 3ES4 KN ∆GE E+S [15.8] ES E+P According to transition state theory, Eqs. [14.7] and [14.14], the rate of the uncatalyzed reaction can be expressed kkBT kkBT ‡ b 3S‡ 4 a b K N 3 S4 [15.9] vN kN 3S4 a h h Similarly, the rate of the enzymatically catalyzed reaction is EP Reaction coordinate FIGURE 15-7 Reaction coordinate diagrams for a hypothetical enzymatically catalyzed reaction involving a single substrate (blue) and the corresponding uncatalyzed reaction (red). See the Animated Figures kkBT kkBT ‡ vE kE 3ES 4 a b 3ES‡ 4 a bKE 3 ES4 [15.10] h h Therefore, combining Eqs. [15.8] to [15.10], ‡ kE KE KT ‡ kN KR KN [15.11] This equation indicates that the more tightly an enzyme binds its reaction’s transition state (KT) relative to the substrate (KR), the greater the rate of the catalyzed reaction (kE) relative to that of the uncatalyzed reaction (kN); that is, catalysis results from the preferential binding and therefore the stabilization of the transition state (S ‡) relative to that of the substrate (S) (Fig. 15-7). According to Eq. [14.15], the ratio of the rates of the catalyzed versus the uncatalyzed reaction is expressed kE ‡ ‡ exp3 1 ¢GN ¢GE 2 RT 4 kN a. Transition State Analogs Are Competitive Inhibitors If an enzyme preferentially binds its transition state, then it can be expected that transition state analogs, stable molecules that resemble S ‡ or one of its components, are potent competitive inhibitors of the enzyme. For example, the reaction catalyzed by proline racemase from Clostridium sticklandii is thought to occur via a planar transition state: _ COO C C N A rate enhancement factor of 106 therefore requires that an enzyme bind its transition state complex with 106-fold higher affinity than its substrate, which corresponds to a 34.2 kJ mol1 stabilization at 25C. This is roughly the free energy of two hydrogen bonds. Consequently, the enzymatic binding of a transition state (ES ‡) by two hydrogen bonds that cannot form in the Michaelis complex (ES) should result in a rate enhancement of 106 based on this effect alone. It is commonly observed that the specificity of an enzyme is manifested by its turnover number (kcat) rather than by its substrate-binding affinity. In other words, an enzyme binds poor substrates, which have a low reaction rate, as well as or even better than good ones, which have a high reaction rate. Such enzymes apparently use a good substrate’s intrinsic binding energy to stabilize the corresponding transition state; that is, a good substrate does not necessarily bind to its enzyme with high affinity, but does so on activation to the transition state. N H + H H [15.12] H + H _ COO H D-Proline L-Proline C _ COO _ N H Planar transition state Proline racemase is competitively inhibited by the planar analogs of proline, pyrrole-2-carboxylate and -1-pyrroline-2-carboxylate, COO– N H Pyrrole-2-carboxylate + COO– N H -1-Pyrroline-2-carboxylate both of which bind to the enzyme with 160-fold greater affinity than does proline. These compounds are therefore thought to be analogs of the transition state in the proline 7884d_c15.qxd 1/23/03 12:28 PM Page 507 mac18 mac18:df_169:7884D: Section 15–2. Lysozyme 6CH2OH ... H O 4 5 O H OH H H O 1 1 CH2OH O H H H CH2OH O H H OH H O CH2OH O H H H O O... H H H 2 3 NH H Lysozyme cleavage 507 C CH3 H NH O O C NH H CH3 C O O NAM C O CH3 O CH3CHCOO– – CH3CHCOO NAG NH H CH3 NAG NAM FIGURE 15-8 The alternating NAG–NAM polysaccharide component of bacterial cell walls. The position of the lysozyme cleavage site is shown. racemase reaction. In contrast, tetrahydrofuran-2-carboxylate, COO– O H Tetrahydrofuran-2-carboxylate which more closely resembles the tetrahedral structure of proline, is not nearly as good an inhibitor as these compounds. A 160-fold increase in binding affinity corresponds, according to Eq. [15.12], to a 12.6 kJ mol1 increase in the free energy of binding. This quantity presumably reflects the additional binding affinity that proline racemase has for proline’s planar transition state over that of the undistorted molecule. Hundreds of transition state analogs for various enzymatic reactions have been reported. Some are naturally occurring antibiotics. Others were designed to investigate the mechanisms of particular enzymes and/or to act as specific enzymatic inhibitors for therapeutic or agricultural use. Indeed, as we discuss in Section 15-4C, the theory that enzymes bind transition states with higher affinity than substrates has led to a rational basis for drug design based on the understanding of specific enzyme reaction mechanisms. susceptible to lysozyme alone has prompted the suggestion that this enzyme mainly helps dispose of bacteria after they have been killed by other means. Hen egg white (HEW) lysozyme is the most widely studied species of lysozyme and is one of the mechanistically best understood enzymes. It is a rather small protein (14.7 kD) whose single polypeptide chain consists of 129 amino acid residues and is internally cross-linked by four disulfide bonds (Fig. 15-9). HEW lysozyme catalyzes the Arg Gly Tyr Ser Asp Asn Tyr Leu Gly 20 Asn Leu Gly His Trp Arg Ala Gln Val Asp Trp Lys Gly Cys S Ala Leu Glu Arg Lys Val In the following two sections, we shall investigate the catalytic mechanisms of several well-characterized enzymes. In doing so, we shall see how enzymes apply the catalytic principles described in Section 15-1. You should note that the great catalytic efficiency of enzymes arises from their simultaneous use of several of these catalytic mechanisms. Lysozyme is an enzyme that destroys bacterial cell walls. It does so, as we saw in Section 11-3B, by hydrolyzing the (1S 4) glycosidic linkages from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) in the alternating NAM–NAG polysaccharide component of cell wall peptidoglycans (Fig. 15-8). It likewise hydrolyzes (1S4)-linked poly(NAG) (chitin), a cell wall component of most fungi. Lysozyme occurs widely in the cells and secretions of vertebrates, where it may function as a bactericidal agent. However, the observation that few pathogenic bacteria are Val Ala Trp Ser Asn Phe Asn Arg Ser Leu Cys S Gly Pro 70 Thr Lys Ile Gly Ala S Leu Leu Cys Ser Asn 90 Ala Thr Ile Gln Ser Asp S S Ala Thr Asn Asn Ser Lys Thr 40 Arg Cys 80 Ile Asn Asn Pro Val Ser Trp Asn Met Ser 100 Val Glu 110 Ala Gly Ala Asn Phe Asp Asp Ala Arg Gly 2 LYSOZYME Ala Lys COO– Arg H3N+ Cys 30 Arg Leu S Gly Phe S Cys 129 Cys 1 S Lys Arg Ala Ala 10 Gly 120 Ile Met Val Thr Asp Arg Cys Asn Trp Thr Trp Asp Arg Gly Ser 60 50 Ser Asn Thr Ile Gln Leu Ile Gly Asp Tyr FIGURE 15-9 Primary structure of HEW lysozyme. The amino acid residues that line the substrate-binding pocket are shown in dark purple. 7884d_c15.qxd 3/24/03 1:01 PM Page 508 mac85 Mac 85:1st shift: 1268_tm:7884d: 508 Chapter 15. Enzymatic Catalysis hydrolysis of its substrate at a rate that is 108-fold greater than that of the uncatalyzed reaction. A. Enzyme Structure The elucidation of an enzyme’s mechanism of action requires a knowledge of the structure of its enzyme–substrate complex. This is because, even if the active site residues have been identified through chemical and physical means, their three-dimensional arrangements relative to the substrate as well as to each other must be known for an understanding of how the enzyme works. However, an enzyme binds its good substrates only transiently before it catalyzes a reaction and releases the products. Consequently, most of our knowledge of enzyme–substrate complexes derives from X-ray studies of enzymes in complex with inhibitors or poor substrates that remain stably bound to the enzyme for the several hours that are usually required to measure a protein crystal’s X-ray diffraction intensities (although techniques for measuring X-ray intensities in less than 1 s have been developed). The large solvent-filled channels that occupy much of the volume of most protein crystals (Section 8-3A) often permit the formation of enzyme–inhibitor complexes by the diffusion of inhibitor molecules into crystals of the native protein. The X-ray structure of HEW lysozyme, which was elucidated by David Phillips in 1965, was the second structure of a protein and the first of an enzyme to be determined at high resolution. The protein molecule is roughly ellipsoidal in shape with dimensions 30 30 45 Å (Fig. 15-10). Its most striking feature is a prominent cleft, the substrate-binding site, that traverses one face of the molecule. The polypeptide chain forms five helical segments as well as a three-stranded antiparallel sheet that comprises much of one wall of the binding cleft (Fig. 15-10b). As expected, most of the nonpolar side chains are in the interior of the molecule, out of contact with the aqueous solvent. ture of the (NAG)3 –lysozyme complex reveals that (NAG)3 is bound on the right side of the enzymatic binding cleft as drawn in Fig. 15-10a for substrate residues A, B, and C. This inhibitor associates with the enzyme through strong hydrogen bonding interactions, some of which involve the acetamido groups of residues A and C, as well as through close-fitting hydrophobic contacts. In an example of induced-fit ligand binding (Section 10-4C), there is a slight (1 Å) closure of lysozyme’s binding cleft on binding (NAG)3. b. Lysozyme’s Catalytic Site Was Identified through Model Building (NAG)3 takes several weeks to hydrolyze under the influence of lysozyme. It is therefore presumed that the complex revealed by X-ray analysis is unproductive; that is, the enzyme’s catalytic site occurs at neither the A¬ B nor the B¬ C bonds. [Presumably, the rare occasions when (NAG)3 hydrolyzes occur when it binds productively at the catalytic site.] In order to locate lysozyme’s catalytic site, Phillips used model building to investigate how a larger substrate could bind to the enzyme. Lysozyme’s active site cleft is long enough to accommodate (NAG)6, which the enzyme rapidly hydrolyzes (Table 15-2). However, the fourth NAG residue (residue D in Fig. 15-10a) appeared unable to bind to the enzyme because its C6 and O6 atoms too closely contact Glu 35, Trp 108, and the acetamido group of NAG residue C. This steric interference could be relieved by distorting the glucose ring from its normal chair conforma- (Opposite) X-Ray structure of HEW lysozyme. (a) The polypeptide chain is shown with a bound (NAG)6 substrate (green). The positions of the backbone C atoms are indicated together with those of the side chains that line the substrate-binding site and form disulfide bonds. The substrate’s sugar rings are designated A, at its nonreducing end (right), through F, at its reducing end (left). Lysozyme catalyzes the hydrolysis of the glycosidic bond between residues D and E. Rings A, B, and C are observed in the X-ray structure of the complex of (NAG)3 with lysozyme; the positions of rings D, E, and F were inferred from model building studies. [Illustration, Irving Geis/Geis Archives Trust. Copyright Howard Hughes Medical Institute. Reproduced with permission.] (b) A ribbon diagram of lysozyme highlighting the protein’s secondary structure and indicating the positions of its catalytically important side chains, Glu 35 and Asp 52 (red). (c) A computer-generated model showing the protein’s molecular envelope ( purple) and C backbone (blue). The side chains of the catalytic residues, Asp 52 (above) and Glu 35 (below), are colored yellow. Note the enzyme’s prominent substrate-binding cleft. [Courtesy of Arthur Olson, The Scripps Research Institute, La Jolla, California.] Parts a, b, and c have See the Interactive approximately the same orientation. FIGURE 15-10 a. The Nature of the Binding Site NAG oligosaccharides of less than five residues are but very slowly hydrolyzed by HEW lysozyme (Table 15-2) although these substrate analogs bind to the enzyme’s active site and are thus its competitive inhibitors. The X-ray struc- Rates of HEW Lysozyme-Catalyzed Hydrolysis of Selected Oligosaccharide Substrate Analogs TABLE 15-2 Compound kcat (s1) (NAG)2 2.5 108 (NAG)3 8.3 106 (NAG)4 6.6 105 (NAG)5 0.033 (NAG)6 0.25 (NAG–NAM)3 0.5 Source: Imoto, T., Johnson, L.N., North, A.C.T., Phillips, D.C., and Rupley, J.A., in Boyer, P.D. (Ed.), The Enzymes (3rd ed.), Vol. 7, p. 842, Academic Press (1972). Exercises and Kinemage Exercise 9 7884d_c15.qxd 3/24/03 1:01 PM Page 509 mac85 Mac 85:1st shift: 1268_tm:7884d: Section 15–2. Lysozyme (a) (b) (c) N Asp 52 Glu 35 C 509 7884d_c15.qxd 3/24/03 1:01 PM Page 510 mac85 Mac 85:1st shift: 1268_tm:7884d: Chapter 15. Enzymatic Catalysis 510 OH tion to that of a half-chair (Fig. 15-11). This distortion, which renders atoms C1, C2, C5, and O5 of residue D coplanar, moves the ¬ C6H2OH group from its normal equatorial position to an axial position where it makes no close contacts and can hydrogen bond to the backbone carbonyl group of Gln 57 and the amido group of Val 109 (Fig. 1512). Continuing the model building, Phillips found that residues E and F apparently bind to the enzyme without distortion and with a number of favorable hydrogen bonding and van der Waals contacts. We are almost in a position to identify lysozyme’s catalytic site. In the enzyme’s natural substrate, every second residue is an NAM. Model building, however, indicated that its lactyl side chain cannot be accommodated in the binding subsites of either residues C or E. Hence, the NAM CH2OH HO O A NAG –O C H3C O N C Asp 101 O H O R H O O CH2 B H NAM O N C H3C O O H NAG NAM A B NAG NAM C D NAG NAM E F ( ) reducing end Trp 62 O CH2 N C Asn 59 N N R D ring in half-chair conformation O Val 109 CH2O D O C NAM H O O O – C C H O Gln 57 Asp 52 O Lysozyme cuts N H O C C O H NH2 O E NAG C C Glu 35 CH2OH O O O N O H3C Asn 44 Ala 107 C CH3 H O5 C5 O H C O H NH2 C4 NAG O Gln 57 Trp 63 N O H H residues must bind to the enzyme in subsites B, D, and F. The observation that lysozyme hydrolyzes (1S4) linkages from NAM to NAG implies that bond cleavage occurs either between residues B and C or between residues D and E. Since (NAG)3 is stably bound to but not cleaved by the enzyme while spanning subsites B and C, the probable cleavage site is between residues D and E. This conclusion is supported by John Rupley’s observation that lysozyme nearly quantitatively hydrolyzes (NAG)6 H O N H3C H C2 C3 Glu C 35 C1 O O Chair conformation R H CH2 F C C4 H3C O C3 C Asn 37 H2N NAM O O N H O5 C O O O C5 Phe 34 O H2N + H2N NH H C2 C1 Half-chair conformation FIGURE 15-11 Chair and half-chair conformations. Hexose rings normally assume the chair conformation. It is postulated, however, that binding by lysozyme distorts the D-ring into the half-chair conformation such that atoms C1, C2, C5, and O5 are See the Animated Figures coplanar. Interactions of lysozyme with its substrate. The view is into the binding cleft with the heavier edges of the rings facing the outside of the enzyme and the lighter ones against the bottom of the cleft. [Illustration, Irving Geis/Geis Archives Trust. Copyright Howard Hughes Medical Institute. Reproduced with permission. Based on an X-ray structure by David Phillips, See Kinemage Oxford University, U.K. PDBid 4LYZ.] FIGURE 15-12 Exercise 9 Arg 114 7884d_c15.qxd 1/23/03 12:28 PM Page 511 mac18 mac18:df_169:7884D: Section 15–2. Lysozyme between the second and third residues from its reducing terminus (the end with a free C1¬OH), just as is expected if the enzyme has six saccharide-binding subsites and cleaves its bound substrate between residues D and E. The bond that lysozyme cleaves was identified by carrying out the lysozyme-catalyzed hydrolysis of (NAG)3 in H218O. The resulting product had 18O bonded to the C1 atom of its newly liberated reducing terminus, thereby demonstrating that bond cleavage occurs between C1 and the bridge oxygen O1: OR H C 511 OR O R + H + H R C + O R H Acetal R ROH R H R + O O C C + R H R Resonance-stabilized carbocation (oxonium ion) H O C1 H O1 C 4 NAc 18 OR OH CH2OH H OH C + H NAc C + H H H OH HO H C OH R lysozyme 18 H H H H2 O O H2O CH2OH Hemiacetal Mechanism of the nonenzymatic acid-catalyzed hydrolysis of an acetal to a hemiacetal. The reaction involves the protonation of one of the acetal’s oxygen atoms followed by cleavage of its C¬ O bond to form an alcohol (ROH) and a resonance-stabilized carbocation (oxonium ion). The addition of water to the oxonium ion forms the hemiacetal and regenerates the H catalyst. Note that the oxonium ion’s C, O, H, R, and R atoms all lie in the same plane. FIGURE 15-13 Thus, lysozyme catalyzes the hydrolysis of the C1 ¬ O1 bond of a bound substrate’s D residue. Moreover, this reaction occurs with retention of configuration, so that the D-ring product remains the anomer. B. Catalytic Mechanism It remains to identify lysozyme’s catalytic groups. The reaction catalyzed by lysozyme, the hydrolysis of a glycoside, is the conversion of an acetal to a hemiacetal. Nonenzymatic acetal hydrolysis is an acid-catalyzed reaction that involves the protonation of a reactant oxygen atom followed by cleavage of its C ¬ O bond (Fig. 15-13). This results in the formation of a resonance-stabilized carbocation that is called an oxonium ion. To attain maximum orbital overlap, and thus resonance stabilization, the oxonium ion’s R and R groups must be coplanar with its C, O, and H atoms (stereoelectronic assistance). The oxonium ion then adds water to yield the hemiacetal and regenerate the acid catalyst. In searching for catalytic groups on an enzyme that mediates acetal hydrolysis, we should therefore seek a potential acid catalyst and possibly a group that could further stabilize an oxonium ion intermediate. a. Glu 35 and Asp 52 Are Lysozyme’s Catalytic Residues The only functional groups in the immediate vicinity of lysozyme’s reaction center that have the required catalytic properties are the side chains of Glu 35 and Asp 52, residues that are invariant in the family of lysozymes of which HEW lysozyme is the prototype. These side chains, which are disposed to either side of the (1S 4) glycosidic linkage to be cleaved (Fig. 15-10), have markedly different environments. Asp 52 is surrounded by several conserved polar residues with which it forms a complex hydrogen bonded network. Asp 52 is therefore predicted to have a normal pK; that is, it should be unprotonated and hence negatively charged throughout the 3 to 8 pH range in which lysozyme is catalytically active. In contrast, the carboxyl group of Glu 35 is nestled in a predominantly nonpolar pocket, where, as we discussed in Section 15-1D, it is likely to remain protonated at unusually high pH’s for carboxyl groups. Indeed, neutron diffraction studies, which provide similar information to X-ray diffraction studies but also reveal the positions of hydrogen atoms, indicate that Glu 35 is protonated at physiological pH’s. The closest approaches in the X-ray structures between the carboxyl O atoms of both Asp 52 and Glu 35 and the C1 ¬ O1 bond of NAG residue D are 3 Å, which makes them the prime candidates for electrostatic and acid catalysts, respectively. 7884d_c15.qxd 512 1/23/03 12:28 PM Page 512 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis b. The Phillips Mechanism With much of the foregoing information, Phillips postulated the following enzymatic mechanism for lysozyme (Fig. 15-14): 1. Lysozyme attaches to a bacterial cell wall by binding to a hexasaccharide unit. In the process, residue D is distorted toward the half-chair conformation in response to the unfavorable contacts that its ¬ C6H2OH group would otherwise make with the protein. Lysozyme, main chain Asp 52 O– NAc 2. Glu 35 transfers its proton to the O1 of the D-ring, the only polar group in its vicinity (general acid catalysis). The C1 ¬ O1 bond is thereby cleaved, generating a resonance-stabilized oxonium ion at C1. 3. The ionized carboxyl group of Asp 52 acts to stabilize the developing oxonium ion through charge–charge interactions (electrostatic catalysis). This carboxylate group apparently cannot form a covalent bond with the substrate because the observed 3 Å distance between C1 and a carboxyl O atom of Asp 52 is much greater than the 1.4 Å length of a C¬ O covalent bond [i.e., the reaction appears to occur via an SN1 (unimolecular nucleophilic substitution) mechanism to yield an oxonium ion, not via a mechanism involving the transient formation of a C ¬ O covalent bond to the enzyme; but see Section 15-2C]. The bond cleavage reaction is facilitated by the strain in the D-ring that distorts it to the planar half-chair conformation. This is a result of the oxonium ion’s required planarity; that is, the initial binding conformation of the D-ring resembles that of the reaction’s transition state (transition state binding catalysis; Fig. 15-15). 4. At this point, the enzyme releases the hydrolyzed E-ring with its attached polysaccharide (the leaving group), yielding a cationic, noncovalent, glycosyl–enzyme intermediate. This oxonium ion subsequently adds H2O from solution in a reversal of the preceding steps to form product and to reprotonate Glu 35. The reaction’s retention of configuration is dictated by the shielding of one of the oxonium ion’s faces by the enzymatic cleft. The enzyme then releases the D-ring product with its attached saccharide, thereby completing the catalytic cycle. D O+ CH2OH O– CH2OH Glu 35 H+ Lysozyme, main chain H2O The Phillips mechanism for the lysozyme reaction. The cleavage of the glycosidic bond between the substrate D- and E-rings occurs through protonation of the bridge oxygen atom by Glu 35. The resulting D-ring oxonium ion is stabilized by the proximity of the Asp 52 carboxylate group and the enzyme-induced distortion of the D-ring. Once the E-ring is released, H2O from solution provides both an OH that combines with the oxonium ion and an H that reprotonates Glu 35. NAc represents the N-acetylamino See Kinemage substituent at C2 of each glucose ring. FIGURE 15-14 Exercise 9 and the Animated Figures CH2OH O H C + H OR H H NHCOCH3 H . O H .. . Glu 35. The mutagenesis of Glu 35 to Gln yields a protein with no detectable catalytic activity (0.1% of wild type), .. a. Identification of the Catalytic Residues Lysozyme’s catalytically important groups have been experimentally identified through site-directed mutagenesis (Section 5-5G) and the use of group-specific reagents: OH– NAc C. Testing the Phillips Mechanism The Phillips mechanism was formulated largely on the basis of structural investigations of lysozyme and a knowledge of the mechanism of nonenzymatic acetal hydrolysis. A variety of evidence has since been gathered that bears on the validity of this mechanism. In the remainder of this section, we discuss the highlights of these studies to illustrate how scientific models evolve. H+ E O CH2OH + O H C H OR H H NHCOCH3 CH3 R = H (NAG) or CH (NAM) _ COO The D-ring oxonium ion intermediate in the Phillips mechanism is stabilized by resonance. This requires that atoms C1, C2, C5, and O5 be coplanar (shading); that is, the hexose ring must assume the half-chair conformation. FIGURE 15-15 7884d_c15.qxd 1/23/03 12:28 PM Page 513 mac18 mac18:df_169:7884D: Section 15–2. Lysozyme although it has only a 1.5-fold decrease in substrate affinity. Glu 35 must therefore be essential for lysozyme’s catalytic activity. Asp 52. The mutagenesis of Asp 52 to Asn, which has a polarity comparable to that of Asp but lacks its negative charge, yields an enzyme with no more than 5% of wildtype lysozyme’s catalytic activity even though this mutation causes an 2-fold increase in the enzyme’s affinity for substrate. Asp 52 is therefore important for enzymatic activity. Noninvolvement of Other Amino Acid Residues. Lysozyme’s other carboxyl groups besides Glu 35 and Asp 52 do not participate in the catalytic process, as was demonstrated by reacting lysozyme with carboxyl-specific reagents in the presence of substrate. This treatment yields an almost fully active enzyme in which all carboxyl groups but Glu 35 and Asp 52 are derivatized. Other group-specific reagents that modify, for instance, His, Lys, Met, or Tyr residues but induce no major protein structure disruptions cause little change in lysozyme’s catalytic efficiency. b. Role of Strain Many of the mechanistic investigations of lysozyme have had the elusive goal of establishing the catalytic role of strain. Not all of these studies, as we shall see, have supported the Phillips mechanism, thereby stimulating a series of investigations that have only recently settled this issue. Measurements of the binding equilibria of various oligosaccharides to lysozyme indicate that all saccharide residues except that binding to the D subsite contribute energetically toward the binding of substrate to lysozyme; binding NAM in the D subsite requires a free energy input of 12 kJ mol1 (Table 15-3). The Phillips mechanism explains this observation as being indicative of the energy penalty of straining the D-ring from its preferred chair conformation toward the half-chair form. As we have discussed in Section 15-1F, an enzyme that catalyzes a reaction by the preferential binding of its transition state has a greater binding affinity for an inhibitor that has the transition state geometry (transition state analog) than it does for its substrate. The -lactone analog of (NAG)4 (Fig. 15-16) is a transition state analog of lysozyme TABLE 15-3 Binding Free Energies of HEW Lysozyme Subsites Site Bound Saccharide Binding Free Energy (kJ mol1) A NAG 7.5 B NAM 12.3 C NAG 23.8 D NAM 12.1 E NAG 7.1 F NAM 7.1 Source: Chipman, D.M. and Sharon, N., Science 165, 459 (1969). H O (NAG)3 CH2OH O H H O OH H H NHCOCH3 O (NAG)3 CH2OH + O H 513 – O OH H H NHCOCH3 The -lactone analog of (NAG)4. Its C1, O1, C2, C5, and O5 atoms are coplanar (shading) because of resonance, as is the D-ring in the reaction intermediate of the Phillips mechanism (compare with Fig. 15-15). FIGURE 15-16 since this compound’s lactone ring has the half-chair conformation that geometrically resembles the proposed oxonium ion transition state of the substrate’s D-ring. X-Ray studies indicate, in accordance with prediction, that this inhibitor binds to lysozyme’s A¬ B¬ C ¬ D subsites such that the lactone ring occupies the D subsite in a halfchairlike conformation. Despite the foregoing, the role of substrate distortion in lysozyme catalysis had been questioned. Theoretical studies by Michael Levitt and Arieh Warshel on substrate binding by lysozyme suggested that the protein is too flexible to mechanically distort the D-ring of a bound substrate. Rather, these calculations implied that transition state stabilization occurs through the displacement by substrate of several tightly bound water molecules from the D subsite. The resulting desolvation of the Asp 52 carboxylate group would significantly enhance its capacity to electrostatically stabilize the transition state oxonium ion. This study therefore concluded that “electrostatic strain” rather than steric strain is the more important factor in stabilizing lysozyme’s transition state. In an effort to obtain further experimental information bearing on the Phillips strain mechanism, Nathan Sharon and David Chipman determined the D subsite–binding affinities of several saccharides by comparing the lysozyme-binding affinities of various substrate analogs. The NAG lactone inhibitor binds to the D subsite with 9.2 kJ mol1 greater affinity than does NAG. This quantity corresponds, according to Eq. [14.15], to no more than an 40-fold rate enhancement of the lysozyme reaction as a result of strain (recall that the difference in binding energy between a transition state analog and a substrate is indicative of the enzyme’s rate enhancement arising from the preferential binding of the transition state complex). Such an enhancement is hardly a major portion of lysozyme’s 108-fold rate enhancement (accounting for only 20% of the reaction’s ¢ ¢G‡cat ; Section 14-1C). Moreover, an N-acetylxylosamine (XylNAc) residue, H O H O H OH OH H H H NHCOCH3 N -Acetylxylosamine residue 7884d_c15.qxd 514 1/23/03 12:28 PM Page 514 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis which lacks the sterically hindered ¬ C6H2OH group of NAM and NAG, has only marginally greater binding affinity for the D subsite (3.8 kJ mol1) than does NAG (2.5 kJ mol1). Yet recall that the Phillips mechanism postulates that it is the unfavorable contacts made by this ¬ C6H 2OH group that promotes D-ring distortion. Nevertheless, lysozyme does not hydrolyze saccharides with XylNAc in the D subsite. The apparent inconsistencies among the foregoing experimental observations were largely rationalized by Michael James’ highly accurate (1.5-Å resolution) X-ray crystal structure determination of lysozyme in complex with NAM–NAG–NAM. This trisaccharide binds, as expected, to the B, C, and D subsites of lysozyme. The NAM in the D subsite, in agreement with the Phillips mechanism, is distorted to the half-chair conformation with its ¬ C6H2OH group in a nearly axial position due to steric clashes that would otherwise occur with the acetamido group of the C subsite NAG (although, contrary to the original Phillips mechanism, Glu 35 and Trp 108 are too far away from the ¬ C6H2OH group to contribute to this distortion). This strained conformation is stabilized by a strong hydrogen bond between the D-ring O6 and the backbone NH of Val 109 (transition state stabilization). Indeed, the mutation of Val 109 to Pro, which lacks the NH group to make such a hydrogen bond, inactivates the enzyme. Lysozyme’s lack of hydrolytic activity when XylNAc occupies its D subsite is likewise explained by the absence of this hydrogen bond and the consequent lesser stability of the XylNAc ring’s half-chair transition state. The unexpectedly small free energy differences in binding NAG, NAG lactone, and XylNAc to the D subsite are explained by the observation that undistorted NAG and XylNAc can be modeled into the D subsite as it occurs in the X-ray structure of the lysozyme NAM–NAG–NAM complex. NAM’s bulky lactyl side chain prevents it from binding to the D subsite in this manner. c. The Lysoyme Reaction Proceeds via a Covalent Intermediate Alternatives to the Phillips mechanism postulate that either (1) the carboxyl group of Asp 52 displaces the leaving group to form a covalent bond to C1, thereby yielding a covalent glycosyl–enzyme ester intermediate that is subsequently displaced by water to yield product (a doubledisplacement mechanism); or (2) water directly displaces the leaving group (a single-displacement mechanism). A single-displacement mechanism would result in inversion of configuration between substrate and product and thus can be ruled out. A double-displacement mechanism would account for the observed retention of configuration in the lysozyme reaction (as does the Phillips mechanism). However, it is at odds with the observation that the distance between C1 in a D subsite–bound saccharide and a carboxyl O of Asp 52 (which participates in a network of hydrogen bonds that apparently hold this side chain in its position) are too long to form a covalent bond (minimally 2.3 Å in the NAM–NAG–NAM complex without significantly disrupting the protein structure vs 1.4 Å for a C¬ O single bond). Indeed, no such covalent bond had been observed in any of the numerous X-ray structures containing hen egg white (HEW) lysozyme. Despite the foregoing, all other -glycosidases of known structure that cleave glycosidic linkages with net retention of configuration at the anomeric carbon (as does HEW lysozyme) have been shown to do so via a covalent glycosyl–enzyme intermediate. The active sites of these socalled retaining -glycosidases structurally resemble that of HEW lysozyme. Moreover, there is no direct evidence indicative of the existence of a long-lived oxonium ion at the active site of any retaining -glycosidase, including HEW lysozyme (the lifetime of a glucosyl oxonium ion in water is 1012 s, a time only slightly longer than that of a bond vibration). Consequently, there had been a growing suspicion that the HEW lysozyme reaction also proceeds via a covalent intermediate, one between the D-ring’s anomeric carbon (C1) and the side chain carboxyl group of Asp 52 to form an ester linkage: H CH2OH O H OR H O O C CH2 Asp 52 H O H NHCOCH3 This intermediate presumably reacts with H2O in what is essentially the reverse of the reaction leading to its formation, thereby yielding the reaction’s second product (a double-displacement mechanism). In this mechanism, the oxonium ion is proposed to be the transition state on the way to forming the covalent intermediate, rather than being an intermediate itself. If, in fact, HEW lysozyme follows this mechanism, the reason that its covalent intermediate had never been observed is that its rate of breakdown must be much faster than its rate of formation. Hence, if this intermediate is to be experimentally observed, its rate of formation must be made significantly greater than its rate of breakdown. To do so, Stephen Withers capitalized on three phenomena. First, if, as postulated, the reaction goes through an oxonium ion transition state, all steps involving its formation should be slowed by the electron withdrawing effects of substituting F (the most electronegative element) at C2 of the D-ring. Second, mutating Glu 35 to Gln (E35Q) removes the general acid–base that catalyzes the reaction, further slowing all steps involving the oxonium ion transition state. Third, substituting an additional F atom at C1 of the D-ring accelerates the formation of the intermediate because this F is a good leaving group. Making all three of these changes should increase the rate of formation of the proposed covalent intermediate relative to its breakdown and hence should result in its accumulation. Withers therefore incubated E35Q HEW lysozyme with NAG-(1S 4)-2-deoxy-2-fluoro--D-glucopyranosyl fluoride (NAG2FGlcF): 7884d_c15.qxd 1/23/03 12:28 PM Page 515 mac18 mac18:df_169:7884D: Section 15–3. Serine Proteases H CH2OH O H OH H HO H O CH2OH O H OH H H H NHCOCH3 F H H F NAG2FGlcF Electrospray ionization mass spectrometry (ESI-MS; Section 7-1J) of this reaction mixture revealed a sharp peak at 14,683 D, consistent with the formation of the proposed covalent intermediate, but no significant peak at or near the 14,314-D molecular mass of the mutant enzyme alone. The X-ray structure of this covalent complex unambiguously reveals the expected 1.4-Å-long covalent bond between C1 of the D-ring NAG and a side chain carboxyl O of Asp 52 (Fig. 15-17). This D-ring NAG adopts an undis- 515 torted chair conformation, thus indicating that it is a reaction intermediate rather than an approximation of the transition state. The superposition of this covalent complex with that of the above described complex of NAM–NAG–NAM with wild-type HEW lysozyme reveals how this covalent bond forms (Fig. 15-17). The shortening of the 3.2-Å distance between the D-ring NAG C1 and the Asp 52 O in the NAM–NAG–NAM complex to 1.4 Å in the covalent complex is almost entirely a consequence of the relaxation of the D-ring from the half-chair to the chair conformation combined with an 45 rotation of the Asp 52 side chain about its C ¬ C bond; the positions of the D-ring O4 and O6 atoms are essentially unchanged. Hence, over 35 years after its formulation, it was shown that the Phillips mechanism must be altered to take into account the transient formation of this covalent glycosyl–enzyme ester intermediate (covalent catalysis). Keep in mind, however, that in order to form this covalent linkage, the D-ring must pass through an oxonium-like transition state, which requires it to transiently assume the half-chair conformation. 3 SERINE PROTEASES Our next example of enzymatic mechanisms is a diverse group of proteolytic enzymes known as the serine proteases (Table 15-4). These enzymes are so named because they have a common catalytic mechanism characterized by the possession of a peculiarly reactive Ser residue that is essential for their enzymatic activity. The serine proteases are the most thoroughly understood family of enzymes as a result of their extensive examination over a nearly 50-year period by kinetic, chemical, physical, and genetic techniques. In this section, we mainly study the best characterized serine proteases, chymotrypsin, trypsin, and elastase. We also consider how these three enzymes, which are synthesized in inactive forms, are physiologically activated. A. Kinetics and Catalytic Groups The HEW lysozyme covalent intermediate. The substrate C- and D-rings and Asp 52 are shown in the superposition of the X-ray structures of the covalent complex formed by reacting E35Q lysozyme with NAG2FGlcF (C green, N blue, O red, and F magenta) and the noncovalent complex of wild-type lysozyme with NAM–NAG–NAM (C yellow, N blue, and O red). Note that the covalent bond between Asp 52 and C1 of the D-ring forms when the D-ring in the noncovalent complex relaxes from its distorted half-chair conformation to an undistorted chair conformation and that the side chain of Asp 52 undergoes an 45 rotation about its C¬ C bond. [Based on X-ray structures by David Vocadlo and Stephen Withers, University of British Columbia, Vancouver, Canada; and Michael James, University of Alberta, Edmonton, Canada. PDBids 1H6M and 9LYZ.] FIGURE 15-17 Chymotrypsin, trypsin, and elastase are digestive enzymes that are synthesized by the pancreatic acinar cells (Fig. 1-10c) and secreted, via the pancreatic duct, into the duodenum (the small intestine’s upper loop). All of these enzymes catalyze the hydrolysis of peptide (amide) bonds but with different specificities for the side chains flanking the scissile (to be cleaved) peptide bond (recall that chymotrypsin is specific for a bulky hydrophobic residue preceding the scissile peptide bond, trypsin is specific for a positively charged residue, and elastase is specific for a small neutral residue; Table 72). Together, they form a potent digestive team. a. Ester Hydrolysis as a Kinetic Model That chymotrypsin can act as an esterase as well as a protease is not particularly surprising since the chemical mechanisms of ester and amide hydrolysis are almost identical. The study of chymotrypsin’s esterase activity has led to important insights concerning this enzyme’s catalytic 7884d_c15.qxd 516 1/23/03 12:28 PM Page 516 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis TABLE 15-4 A Selection of Serine Proteases Enzyme Source Function Trypsin Pancreas Digestion of proteins Chymotrypsin Pancreas Digestion of proteins Elastase Pancreas Digestion of proteins Thrombin Vertebrate serum Blood clotting Plasmin Vertebrate serum Dissolution of blood clots Kallikrein Blood and tissues Control of blood flow Complement C1 Serum Cell lysis in the immune response Acrosomal protease Sperm acrosome Penetration of ovum Lysosomal protease Animal cells Cell protein turnover Cocoonase Moth larvae Dissolution of cocoon after metamorphosis -Lytic protease Bacillus sorangium Possibly digestion Proteases A and B Streptomyces griseus Possibly digestion Subtilisin Bacillus subtilis Possibly digestion Source: Stroud, R.M., Sci. Am. 231(1), 86 (1974). Time course of p-nitrophenylacetate hydrolysis as catalyzed by two different concentrations of chymotrypsin. The enzyme rapidly binds substrate and releases the first product, p-nitrophenolate ion, but the second product, acetate ion, is released more slowly. Consequently, the rate of p-nitrophenolate generation begins rapidly (burst phase) but slows as acyl–enzyme complex accumulates until the rate of p-nitrophenolate generation approaches that of acetate release (steady state). The extrapolation of the steady state curve to zero time (dashed lines) indicates the initial concentration of active enzyme. [After Hartley, B.S. and Kilby, B.A., Biochem. J. 56, 294 (1954).] 4 FIGURE 15-18 [ p-Nitrophenolate] (mM) Burst phase Steady state phase L–1 g⋅m 0.8 m 3 2 –1 0.4 mg ⋅ mL 1 0 2 4 6 Time (min) mechanism. Kinetic measurements by Brian Hartley of the chymotrypsin-catalyzed hydrolysis of p-nitrophenylacetate O CH3 C NO2 O 8 10 12 p-nitrophenolate ion forming a covalent acyl–enzyme intermediate that (2) is slowly hydrolyzed to release acetate: O p-Nitrophenylacetate H2O chymotrypsin 2H+ CH3 C O NO2 p-Nitrophenylacetate + Chymotrypsin O CH3 C Acetate O– + –O NO2 –O fast O indicated that the reaction occurs in two phases (Fig. 15-18): 1. The “burst phase,” in which the highly colored p-nitrophenolate ion is rapidly formed in amounts stoichiometric with the quantity of active enzyme present. 2. The “steady-state phase,” in which p-nitrophenolate is generated at a reduced but constant rate that is independent of substrate concentration. These observations have been interpreted in terms of a two-stage reaction sequence in which the enzyme (1) rapidly reacts with the p-nitrophenylacetate to release NO2 p-Nitrophenolate p-Nitrophenolate CH3 C Enzyme Acyl–enzyme intermediate H2O slow H+ O CH3 C O– Acetate + Enzyme Enzyme 7884d_c15.qxd 1/23/03 12:28 PM Page 517 mac18 mac18:df_169:7884D: Section 15–3. Serine Proteases Chymotrypsin evidently follows a Ping Pong Bi Bi mechanism (Section 14-5A). Chymotrypsin-catalyzed amide hydrolysis has been shown to follow a reaction pathway similar to that of ester hydrolysis but with the first step of the reaction, enzyme acylation, being rate determining rather than the deacylation step. types of nerve cells (Sections 12-4D and 20-5C). The inactivation of acetylcholinesterase prevents the otherwise rapid hydrolysis of the acetylcholine released by a nerve impulse and thereby interferes with the regular sequence of nerve impulses. DIPF is of such great toxicity to humans that it has been used militarily as a nerve gas. Related compounds, such as parathion and malathion, b. Identification of the Catalytic Residues Chymotrypsin’s catalytically important groups were identified by chemical labeling studies. These are described below. O O 2N Ser 195. A diagnostic test for the presence of the active Ser of serine proteases is its reaction with diisopropylphosphofluoridate (DIPF): + F CH3 O CH2 CH3 O Diisopropylphosphofluoridate (DIPF) CH(CH3)2 O O P O CH2CH3 CH CH2 C O S CH2 O CH3 P S O CH3 are useful insecticides because they are far more toxic to insects than to mammals. CH(CH3)2 CH2 S Malathion O (Active Ser) P C O P CH2CH3 O O CH2OH O Parathion CH(CH3)2 (Active Ser) 517 + O HF O His 57. A second catalytically important residue was discovered through affinity labeling. In this technique, a substrate analog bearing a reactive group specifically binds at the enzyme’s active site, where it reacts to form a stable covalent bond with a nearby susceptible group (these reactive substrate analogs have therefore been described as the “Trojan horses” of biochemistry). The affinity labeled groups can subsequently be identified by peptide mapping (Section 7-1K). Chymotrypsin specifically binds tosyl-L-phenylalanine chloromethyl ketone (TPCK), CH(CH3)2 DIP–Enzyme which irreversibly inactivates the enzyme. Other Ser residues, including those on the same protein, do not react with DIPF. DIPF reacts only with Ser 195 of chymotrypsin, thereby demonstrating that this residue is the enzyme’s active Ser. The use of DIPF as an enzyme inactivating agent came about through the discovery that organophosphorus compounds such as DIPF are potent nerve poisons. The neurotoxicity of DIPF arises from its ability to inactivate acetylcholinesterase, a serine esterase that catalyzes the hydrolysis of acetylcholine: O + (CH3)3N CH2 CH2 O O CH3 + CH3 C H2O CH2 Choline OH + H CH2 C CH3 Acetylcholine is a neurotransmitter: It transmits nerve impulses across the synapses (junctions) between certain CH2Cl Cl HCl CH2 N CH2 N O –O C Chymotrypsin N O CH O Chymotrypsin acetylcholinesterase CH2 NH because of its resemblance to a Phe residue (one of chymotrypsin’s preferred residues; Table 7-2). Active site– bound TPCK’s chloromethyl ketone group is a strong alkylating agent; it reacts with His 57 (Fig. 15-19), thereby Acetylcholine + (CH3)3N S CH2 O His 57 + C R TPCK O N CH2 C O R FIGURE 15-19 alkylate His 57. Reaction of TPCK with chymotrypsin to 7884d_c15.qxd 3/24/03 1:01 PM Page 518 mac85 Mac 85:1st shift: 1268_tm:7884d: 518 Chapter 15. Enzymatic Catalysis (a) (a) X-Ray structure of bovine trypsin. (a) A drawing of the enzyme in complex with a polypeptide substrate (green) that has its Arg side chain occupying the enzyme’s specificity pocket (stippling). The C backbone of the enzyme is shown together with its disulfide bonds and the side chains of the catalytic triad, Ser 195, His 57, and Asp 102. The active sites of chymotrypsin and elastase contain almost identically arranged catalytic triads. [Illustration, Irving Geis/Geis Archives Trust. Copyright Howard Hughes Medical Institute. FIGURE 15-20 Reproduced with permission.] (b) A ribbon diagram of trypsin highlighting its secondary structure and indicating the arrangement of its catalytic triad. (c) A drawing showing the surface of trypsin (blue) superimposed on its polypeptide backbone ( purple). The side chains of the catalytic triad are shown in green. [Courtesy of Arthur Olson, The Scripps Research Institute, La Jolla, California.] Parts a, b, and c have See Kinemage approximately the same orientation. Exercise 10-1 7884d_c15.qxd 3/24/03 1:01 PM Page 519 mac85 Mac 85:1st shift: 1268_tm:7884d: Section 15–3. Serine Proteases amino acid residue numbering scheme. Bovine chymotrypsin is synthesized as an inactive 245-residue precursor named chymotrypsinogen that is proteolytically converted to chymotrypsin (Section 15-3E). In what follows, the numbering of the amino acid residues in chymotrypsin, trypsin, and elastase will be that of the corresponding residues in bovine chymotrypsinogen. The X-ray structure of bovine chymotrypsin was elucidated in 1967 by David Blow. This was followed by the determination of the structures of bovine trypsin (Fig. 15-20) by Robert Stroud and Richard Dickerson, and porcine elastase by David Shotton and Herman Watson. Each of these proteins is folded into two domains, both of which have extensive regions of antiparallel -sheets in a barrellike arrangement but contain little helix. The catalytically essential His 57 and Ser 195 are located at the substratebinding site together with the invariant (in all serine proteases) Asp 102, which is buried in a solvent-inaccessible pocket. These three residues form a hydrogen bonded constellation referred to as the catalytic triad (Figs. 15-20 and 15-21). inactivating the enzyme. The TPCK reaction is inhibited by -phenylpropionate, CH2 CH2 COO– -Phenylpropionate a competitive inhibitor of chymotrypsin that presumably competes with TPCK for its enzymatic binding site. Moreover, the TPCK reaction does not occur in 8M urea, a denaturing reagent, or with DIP–chymotrypsin, in which the active site is blocked. These observations establish that His 57 is an essential active site residue of chymotrypsin. B. X-Ray Structures Bovine chymotrypsin, bovine trypsin, and porcine elastase are strikingly homologous: The primary structures of these 240-residue monomeric enzymes are 40% identical and their internal sequences are even more alike (in comparison, the and chains of human hemoglobin have a 44% sequence identity). Furthermore, all of these enzymes have an active Ser and a catalytically essential His as well as similar kinetic mechanisms. It therefore came as no surprise when their X-ray structures all proved to be closely related. To most conveniently compare the structures of these three digestive enzymes, they have been assigned the same a. The Structural Basis of Substrate Specificity Can Be Quite Complex The X-ray structures of the above three enzymes suggest the basis for their differing substrate specificities (Table 7-2): 1. In chymotrypsin, the bulky aromatic side chain of the preferred Phe, Trp, or Tyr residue that contributes the C His 57 Ser 195 N Asp 102 (b) FIGURE 15-20 (c) (Continued) 519 7884d_c15.qxd 520 1/23/03 12:28 PM Page 520 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis Gly 193 Ser 195 Catalytic triad Asp 194 His 57 Ile 16 Asp 102 carbonyl group of the scissile peptide fits snugly into a slitlike hydrophobic pocket, the specificity pocket, that is located near the catalytic groups (Fig. 15-20a). 2. In trypsin, the residue corresponding to chymotrypsin Ser 189, which lies at the back of the specificity pocket, is the anionic residue Asp. The cationic side chains of trypsin’s preferred residues, Arg or Lys, can therefore form ion pairs with this Asp residue. The rest of chymotrypsin’s specificity pocket is preserved in trypsin so that it can accommodate the bulky side chains of Arg and Lys. 3. Elastase is so named because it rapidly hydrolyzes the otherwise nearly indigestible Ala, Gly, and Val-rich protein elastin (a connective tissue protein with rubberlike elastic properties). Elastase’s specificity pocket is largely occluded by the side chains of a Val and a Thr residue that replace two Gly’s lining this pocket in both chymotrypsin and trypsin. Consequently elastase, whose specificity pocket is better described as a depression, specifically cleaves peptide bonds after small neutral residues, particularly Ala. In contrast, chymotrypsin and trypsin hydrolyze such peptide bonds extremely slowly because these small substrates cannot be sufficiently immobilized on the enzyme surface for efficient catalysis to occur (Section 15-1E). Thus, for example, trypsin catalyzes the hydrolysis of peptidyl amide substrates with an Arg or Lys residue preceding the scissile bond with an efficiency, as measured by kcatKM (Section 14-2B), that is 106-fold greater than that for the corresponding Phe-containing substrates. Conversely, chymotrypsin catalyzes the hydrolysis of substrates after Phe, Trp, and Tyr residues 104-fold more efficiently than after the corresponding Lys-containing substrates. Despite the foregoing, the mutagenic change in trypsin of Asp 189 S Ser (D189S) by William Rutter did not switch its specificity to that of chymotrypsin but instead yielded FIGURE 15-21 The active site residues of chymotrypsin. The view is in approximately the same direction as in Fig. 15-20. The catalytic triad consists of Ser 195, His 57, and Asp 102. [After Blow, D.M. and Steitz, T.A., Annu. Rev. Biochem. 39, 86 (1970).] a poor, nonspecific protease. Moreover, even replacing the other three residues in trypsin’s specificity pocket that differ from those in chymotrypsin, with those of chymotrypsin, fails to yield a significantly improved enzyme. However, trypsin is converted to a reasonably active chymotrypsin-like enzyme when, in addition to the foregoing changes (collectively designated S1), both of its two surface loops that connect the walls of the specificity pocket, L1 (residues 185–188) and L2 (residues 221–225), are replaced by those of chymotrypsin (termed TrSCh[S1L1L2]). Although this mutant enzyme still has a low substrate-binding affinity, KS, the additional mutation Y172W in a third surface loop yields an enzyme (TrSCh[S1L1L2+Y172W]) that has 15% of chymotrypsin’s catalytic efficiency. Curiously, these loops, whose sequences are largely conserved in each enzyme, are not structural components of either the specificity pocket or the extended substrate binding site in chymotrypsin or in trypsin (Fig. 15-20a). Careful comparisons, by Charles Craik and Robert Fletterick, of the X-ray structures of chymotrypsin and trypsin with those of the closely similar TrSCh[S1L1 L2] and TrSCh[S1L1L2Y172W] in complex with a Phe-containing chloromethyl ketone inhibitor reveal the structural basis of substrate specificity in trypsin and chymotrypsin. Efficient catalysis in the serine proteases requires that the enzyme’s active site be structurally intact and that the substrate’s scissile bond be properly positioned relative to the catalytic triad and other components of the active site (see below). The above mutagenic changes do not affect the structure of the catalytic triad or those portions of the active site that bind the substrate’s leaving group (that segment on the C-terminal side of the scissile bond). However, the main chain conformation of the conserved Gly 216 (which forms two hydrogen bonds to the backbone of the third residue before the substrate’s scis- 7884d_c15.qxd 2/10/03 9:25 AM Page 521 mac18 mac18:df_169:7884D: Section 15–3. Serine Proteases sile bond in an antiparallel pleated sheet–like arrangement) differs in trypsin and chymotrypsin and adopts a chymotrypsin-like structure in both hybrid proteins. Evidently, if Gly 216 adopts a trypsin-like conformation, the scissile bond in Phe-containing substrates is misoriented for efficient catalysis. Thus, despite the fact that Gly 216 is conserved in trypsin and chymotrypsin, the differing structures of loop L2 in the two enzymes maintain it in distinct conformations. Loop L1, which interacts with L2 in both trypsin and chymotrypsin, is largely disordered in the X-ray structure of TrSCh[S1L1L2]. Modeling a trypsin-like L1 into TrSCh[S1L1L2] results in severe steric clashes with the chymotrypsin-like L2. Thus, the requirement of a chymotrypsin-like L1 for the efficient catalysis by TrS Ch[S1L1L2] appears to arise from the need to permit L2 to adopt a chymotrypsin-like conformation. Residue 172 is located at the base of the specificity pocket. The improvement in substrate binding affinity of TrSCh[S1L1L2Y172W] over TrSCh[S1L1L2] arises from structural rearrangements in this region of the enzyme caused by the increased bulk and different hydrogen bonding requirements of Trp versus Tyr. These changes appear to improve both the structural stability of residues forming the specificity pocket and their specificity for chymotrypsin-like substrates. These results therefore highlight an important caveat for genetic engineers: Enzymes are so exquisitely tailored to their functions that they often respond to mutagenic tinkering in unexpected ways. b. Evolutionary Relationships among Serine Proteases We have seen that sequence and structural homologies among proteins reveal their evolutionary relationships (Sections 7-3 and 9-6). The great similarities among chymotrypsin, trypsin, and elastase indicate that these proteins evolved through gene duplications of an ancestral serine protease followed by the divergent evolution of the resulting enzymes (Section 7-3C). Several serine proteases from various sources provide further insights into the evolutionary relationships among the serine proteases. Streptomyces griseus protease A (SGPA) is a bacterial serine protease of chymotryptic specificity that exhibits extensive structural similarity, although only 20% sequence identity, with the pancreatic serine proteases. The primordial trypsin gene evidently arose before the divergence of prokaryotes and eukaryotes. There are three known serine proteases whose primary and tertiary structures bear no discernible relationship to each other or to chymotrypsin but which, nevertheless, contain catalytic triads at their active sites whose structures closely resemble that of chymotrypsin: Subtilisin NH+3 521 ClpP Serine Chymotrypsin carboxypeptidase II protease NH+3 NH+3 NH+3 Asp 32 His 64 His 57 Ser 146 Asp 102 Ser 125 Leu 126 Gly 127 Ser 97 Asp 338 Ser 195 Ser 221 Ser 214 Trp 215 Gly 216 COO– COO– His 397 COO– His 122 Asp 171 COO– Relative positions of the active site residues in subtilisin, chymotrypsin, serine carboxypeptidase II, and ClpP protease. The peptide backbones of Ser 214, Trp 215, and Gly 216 in chymotrypsin, and their counterparts in subtilisin, participate in substrate-binding interactions. [After Robertus, J.D., Alden, R.A., Birktoft, J.J., Kraut, J., Powers, J.C., and See Kinemage Wilcox, P.E., Biochemistry 11, 2449 (1972).] FIGURE 15-22 Exercise 10-2 carboxypeptidase A (Fig. 8-19a) even though the latter protease has an entirely different catalytic mechanism from that of the serine proteases (see Problem 3). 3. E. coli ClpP, which functions in the degradation of cellular proteins (Section 32-6B). Since the orders of the corresponding active site residues in the amino acid sequences of the four types of serine proteases are quite different (Fig. 15-22), it seems highly improbable that they could have evolved from a common ancestor serine protease. These proteins apparently constitute a remarkable example of convergent evolution: Nature seems to have independently discovered the same catalytic mechanism at least four times. (In addition, human cytomegalovirus protease, an essential protein for virus replication that bears no resemblance to the above proteases, has active site Ser and His residues whose relative positions are similar to those in other serine proteases but lacks an active site Asp residue; it appears to have a catalytic dyad.) C. Catalytic Mechanism 1. Subtilisin, an endopeptidase that was originally isolated from Bacillus subtilis. See Guided Exploration 12: The Catalytic Mechanism of Serine Proteases The extensive active site homologies among the 2. Wheat germ serine carboxypeptidase II, an exopeptidase whose structure is surprisingly similar to that of various serine proteases indicate that they all have the same catalytic mechanism. On the basis of considerable 7884d_c15.qxd 1/23/03 12:28 PM Page 522 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis 522 1. After chymotrypsin has bound substrate to form the Michaelis complex, Ser 195, in the reaction’s rate-determining step, nucleophilically attacks the scissile peptide’s carbonyl group to form a complex known as the tetrahedral chemical and structural data gathered in many laboratories, the following catalytic mechanism has been formulated for the serine proteases, here given in terms of chymotrypsin (Fig. 15-23): His 57 Asp 102 H 2C CH2 H .... N1 O .. CH2 H O 3 N+ 1 C O– H Tetrahedral intermediate 2 His 57 Asp 102 His 57 CH2 H 2C Ser 195 N1 CH2 .O ... H C – .... .... H O R N Enzyme–substrate complex .O H2C ... C – O R O H CH2 H Nucleophilic attack C N Asp 102 Ser 195 N1 R R Substrate polypeptide H O 3 N CH2 .O – C ... H 2C Ser 195 .... .O C – ... His 57 Asp 102 H2O Ser 195 N1 O 3 2 3 CH2 N CH2 N O H R H R R C N C O O H New N-terminus of cleaved polypeptide chain RNH2 H O O Acyl–enzyme intermediate 3 . .... C – O CH2 H 2C H Ser 195 N N+ Ser 195 H N CH2 N CH2 H O H R O C H O– Tetrahedral intermediate Catalytic mechanism of the serine proteases. The reaction involves (1) the nucleophilic attack of the active site Ser on the carbonyl carbon atom of the scissile peptide bond to form the tetrahedral intermediate; (2) the decomposition of the tetrahedral intermediate to the acyl– enzyme intermediate through general acid catalysis by the FIGURE 15-23 C – O 4 CH2 .O ... .... H2C O ... His 57 Asp 102 His 57 Asp 102 O + New C-terminus of cleaved polypeptide chain O H R C O Active enzyme active site Asp-polarized His, followed by loss of the amine product and its replacement by a water molecule; (3) the reversal of Step 2 to form a second tetrahedral intermediate; and (4) the reversal of Step 1 to yield the reaction’s carboxyl product and the active enzyme. 7884d_c15.qxd 3/24/03 1:01 PM Page 523 mac85 Mac 85:1st shift: 1268_tm:7884d: Section 15–3. Serine Proteases intermediate (covalent catalysis). X-Ray studies indicate that Ser 195 is ideally positioned to carry out this nucleophilic attack (proximity and orientation effects). The imidazole ring of His 57 takes up the liberated proton, thereby forming an imidazolium ion (general base catalysis). This process is aided by the polarizing effect of the unsolvated carboxylate ion of Asp 102, which is hydrogen bonded to His 57 (electrostatic catalysis; see Section 15-3D). Indeed, the mutagenic replacement of trypsin’s Asp 102 by Asn leaves the enzyme’s KM substantially unchanged at neutral pH but reduces its kcat to 0.05% of its wild-type value. Neutron diffraction studies have demonstrated that Asp 102 remains a carboxylate ion rather than abstracting a proton from the imidazolium ion to form an uncharged carboxylic acid group. The tetrahedral intermediate has a well-defined, although transient, existence. We shall see that much of chymotrypsin’s catalytic power derives from its preferential binding of the transition state leading to this intermediate (transition state binding catalysis). 523 The portion of BPTI in contact with the trypsin active site resembles bound substrate. The side chain of BPTI Lys 15I (here “I” differentiates BPTI residues from trypsin residues) occupies the trypsin specificity pocket (Fig. 15-24a) and the peptide bond between Lys 15I and Ala 16I is positioned as if it were the scissile peptide bond (Fig. 15-24b). What is most remarkable about this structure is that its active site complex assumes a conformation well along the reaction coordinate toward the tetrahedral intermediate: The side chain oxygen of trypsin Ser 195, the active Ser, is in closer-than-van der Waals contact (2.6 Å) with the pyramidally distorted carbonyl carbon of BPTI’s “scissile” peptide. Despite this close contact, the proteolytic reaction cannot proceed past this point along the reaction coordinate because of the rigidity of the active site complex and because it is so tightly sealed that the leaving group cannot leave and water cannot enter the reaction site. Protease inhibitors are common in nature, where they have protective and regulatory functions. For example, certain 2. The tetrahedral intermediate decomposes to the acyl–enzyme intermediate under the driving force of proton donation from N3 of His 57 (general acid catalysis). The amine leaving group (RNH2, the new N-terminal portion of the cleaved polypeptide chain) is released from the enzyme and replaced by water from the solvent. 3 & 4. The acyl-enzyme intermediate (which, in the absence of enzyme, would be a stable compound) is rapidly deacylated by what is essentially the reverse of the previous steps followed by the release of the resulting carboxylate product (the new C-terminal portion of the cleaved polypeptide chain), thereby regenerating the active enzyme. In this process, water is the attacking nucleophile and Ser 195 is the leaving group. D. Testing the Catalytic Mechanism The formulation of the foregoing model for catalysis by serine proteases has prompted numerous investigations of its validity. In this section we discuss several of the most revealing of these studies. (a) Ser 195 O H Ala 16I O a. The Tetrahedral Intermediate Is Mimicked in a Complex of Trypsin with Trypsin Inhibitor Convincing structural evidence for the existence of the tetrahedral intermediate was provided by Robert Huber in an X-ray study of the complex between bovine pancreatic trypsin inhibitor (BPTI) and trypsin. The 58-residue protein BPTI, whose folding pathway we examined in Section 9-1C, binds to and inactivates trypsin, thereby preventing any trypsin that is prematurely activated in the pancreas from digesting that organ (see Section 15-3E). BPTI binds to the active site region of trypsin across a tightly packed interface that is cross-linked by a complex network of hydrogen bonds. This complex’s 1013M1 association constant, among the largest of any known protein–protein interaction, emphasizes BPTI’s physiological importance. C C C (b) (b) N H Lys 15I FIGURE 15-24 Trypsin–BPTI complex. (a) The X-ray structure shown as a cutaway surface drawing indicating how trypsin (red) binds BPTI (green). The green protrusion extending into the red cavity near the center of the figure represents the Lys 15I side chain occupying trypsin’s specificity pocket. Note the close complementary fit of these two proteins. [Courtesy of Michael Connolly, New York University.] (b) Trypsin Ser 195, the active Ser, is in closer-than-van der Waals contact with the carbonyl carbon of BPTI’s scissile peptide, which is pyramidally distorted toward Ser 195. The normal proteolytic reaction is apparently arrested somewhere along the reaction coordinate between the Michaelis complex and the tetrahedral intermediate. 7884d_c15.qxd 524 1/23/03 12:28 PM Page 524 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis plants release protease inhibitors in response to insect bites, thereby causing the offending insect to starve by inactivating its digestive enzymes. Protease inhibitors constitute 10% of the nearly 200 proteins of blood serum. For instance, 1-proteinase inhibitor, which is secreted by the liver, inhibits leukocyte elastase (leukocytes are a type of white blood cell; the action of leukocyte elastase is thought to be part of the inflammatory process). Pathological variants of 1-proteinase inhibitor with reduced activity are associated with pulmonary emphysema, a degenerative disease of the lungs resulting from the hydrolysis of its elastic fibers. Smokers also suffer from reduced activity of their 1-proteinase inhibitor because of the oxidation of its active site Met residue. Full activity of this inhibitor is not regained until several hours after smoking. b. Serine Proteases Preferentially Bind the Transition State Detailed comparisons of the X-ray structures of several serine protease–inhibitor complexes have revealed a further structural basis for catalysis in these enzymes (Fig. 15-25): 1. The conformational distortion that occurs with the formation of the tetrahedral intermediate causes the carbonyl oxygen of the scissile peptide to move deeper into the active site so as to occupy a previously unoccupied position, the oxyanion hole. 2. There it forms two hydrogen bonds with the enzyme that cannot form when the carbonyl group is in its normal trigonal conformation. These two enzymatic hydrogen bond donors were first noted by Joseph Kraut to occupy corresponding positions in chymotrypsin and subtilisin. He proposed the existence of the oxyanion hole based on the premise that convergent evolution had made the active sites of these unrelated enzymes functionally identical. 3. The tetrahedral distortion, moreover, permits the formation of an otherwise unsatisfied hydrogen bond between the enzyme and the backbone NH group of the residue preceding the scissile peptide. Consequently, the enzyme binds the tetrahedral intermediate in preference to either the Michaelis complex or the acyl–enzyme intermediate. It is this phenomenon that is responsible for much of the catalytic efficiency of serine proteases (see below). In fact, the reason that DIPF is such an effective inhibitor of serine proteases is because its tetrahedral phosphate group makes this compound a transition state analog of the enzyme. c. The Tetrahedral Intermediate and the Water Molecule Attacking the Acyl–Enzyme Intermediate Have Been Directly Observed Most enzymatic reactions turn over far too rapidly for their intermediate states to be studied by X-ray or NMR techniques. Consequently, much of our structural knowledge of these intermediate states derives from the study of enzyme–inhibitor complexes or complexes of substrates with inactivated enzymes. Yet the structural relevance of these complexes is subject to doubt precisely because they are catalytically unproductive. In an effort to rectify this situation for serine proteases, Janos Hadju and Christopher Schofield searched for peptide–protease complexes that are stable at pH’s at which the protease is inactive but which could be rendered active by changing the pH. To do so, they screened libraries (b) (a) Oxyanion hole Ser 195 Ser 195 His 57 N H O Cα Cβ . C R ... O H O NH Gly 193 .... Gly 193 HN N O NH ...–O C Asp 102 R′ NH ... O– R N H O C Gly 193 Gly 193 Transition state stabilization in the serine proteases. (a) In the Michaelis complex, the trigonal carbonyl carbon of the scissile peptide is conformationally constrained from binding in the oxyanion hole (upper left). (b) In the tetrahedral intermediate, the now charged carbonyl oxygen of the scissile peptide (the oxyanion) has entered the oxyanion hole, thereby hydrogen bonding to the backbone NH groups of O O C C FIGURE 15-25 His 57 N H ... N H Cα HN ... H Cβ –O N + N..H.. C Asp 102 R′ Gly 193 and Ser 195. The consequent conformational distortion permits the NH group of the residue preceding the scissile peptide bond to form an otherwise unsatisfied hydrogen bond to Gly 193. Serine proteases therefore preferentially bind the tetrahedral intermediate. [After Robertus, J.D., Kraut, J., Alden, R.A., and Birktoft, J.J., Biochemistry 11, 4302 (1972).] See Kinemage Exercise 10-3 7884d_c15.qxd 3/24/03 1:01 PM Page 525 mac85 Mac 85:1st shift: 1268_tm:7884d: Section 15–3. Serine Proteases 525 of peptides for their ability to bind to porcine pancreatic elastase at pH 3.5 (at which pH His 57 is protonated and hence unable to act as a general base) through the use of ESI-MS (Section 7-1J). They thereby discovered that YPFVEPI, a heptapeptide segment of the human milk protein -casein that is named BCM7, forms a complex with elastase, whose mass is consistent with the formation of an ester linkage between BCM7 and the enzyme. In the presence of 18OH2 at pH 7.5 (where elastase is active), the 18O label was incorporated into both BCM7 and the elastase– BCM7 complex, thereby demonstrating that the reaction of BCM7 with elastase is reversible at this pH. Fragmentation studies by fast atom bombardment–tandem mass spectroscopy (FAB–MS/MS; Section 7-1J) further revealed that BCM7 that had been incubated with elastase in the presence of 18OH2 at pH 7.5 incorporated the 18O label into only its C-terminal Ile residue. The X-ray structure of the BCM7–elastase complex at pH 5 (Fig. 15-26a) revealed that BCM7’s C-terminal carboxyl group, in fact, forms an ester linkage with elastase’s Ser 195 side chain hydroxyl group to form the expected acyl–enzyme intermediate. Moreover, this X-ray structure reveals the presence of a bound water molecule that appears poised to nucleophilically attack the ester linkage (the distance from this water molecule to BCM7’s Cterminal C atom is 3.1 Å and the line between them is nearly perpendicular to the plane of the acyl group). His 57, which is hydrogen bonded to this water molecule, is properly positioned to abstract one of its protons, thereby activating it for the nucleophilic attack (general base catalysis). The carbonyl O atom of the acyl group occupies the enzyme’s oxyanion hole such that it is hydrogen bonded to the main chain N atoms of both Ser 195 and Gly 193. This is in agreement with spectroscopic measurements indicating that the acyl–enzyme intermediate’s carbonyl group is, in fact, hydrogen bonded to the oxyanion hole. It was initially assumed that the oxyanion hole acts only to stabilize the tetrahedral oxyanion transition state that resides near the tetrahedral intermediate on the catalytic reaction coordinate. However, it now appears that the oxyanion hole also functions to polarize the carbonyl group of the acyl–enzyme intermediate toward an oxyanion (electrostatic catalysis). The catalytic reaction was initiated in crystals of the BCM7–elastase complex by transferring them to a buffer at pH 9. After soaking in this buffer for 1 min, the crystals were rapidly frozen in liquid N2 (196°C), thereby arresting the enzymatic reaction (recall that the catalytically essential collective motions of proteins cease at such low temperatures; Section 9-4). The X-ray structure of such a frozen crystal (Fig. 15-26b) revealed that the above acyl– enzyme intermediate had converted to the tetrahedral in- (a) (b) X-Ray structures of porcine pancreatic elastase in complex with the heptapeptide BCM7 (YPFVEPI). The residues of elastase are specified by the three-letter code and those of BCM7 are specified by the one-letter code. (a) The complex at pH 5. The enzyme’s active site residues and the heptapeptide (whose N-terminal three residues are disordered) are shown in ball-and-stick form with elastase C green, BCM7 C cyan, N blue, O red, S yellow, and the bond between the Ser 195 O atom and the C-terminal C atom of BCM7 magenta. The enzyme-bound water molecule, which appears poised to nucleophilically attack the acyl–enzyme’s carbonyl C atom, is represented by an orange sphere. The dashed gray lines represent the catalytically important hydrogen bonds and the dotted gray line indicates the trajectory that the bound water molecule presumably follows in nucleophilically attacking the acyl group’s carbonyl C atom. (b) The complex after being brought to pH 9 for 1 min and then rapidly frozen in liquid nitrogen. The various groups in the structure are represented and colored as in Part a. Note that the water molecule in Part a has become a hydroxyl substituent (orange) to the carbonyl C atom, thereby yielding the tetrahedral intermediate. [Based on X-ray structures by Christopher Schofield and Janos Hadju, University of Oxford, U.K. PDBids (a) 1HAX and (b) 1HAZ.] FIGURE 15-26 7884d_c15.qxd 526 1/23/03 12:28 PM Page 526 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis termediate, whose oxyanion, as expected, remained hydrogen bonded to the N atoms of Ser 195 and Gly 193. Comparison of this crystal structure with that of the acyl– enzyme intermediate reveals that the enzyme’s active site residues do not significantly change their positions in the conversion from the acyl–enzyme intermediate to the tetrahedral intermediate. However, the peptide substrate must do so out of steric necessity when the trigonal planar acyl group converts to the tetrahedral oxyanion (compare Figs. 15-26a and 15-26b). In response, several enzyme residues that contact the peptide but which are distant from the active site also shift their positions (not shown in Fig. 15-26). d. The Role of the Catalytic Triad: Low-Barrier Hydrogen Bonds The earlier literature postulated that the Asp 102polarized His 57 side chain directly abstracts a proton from Ser 195, thereby converting its weakly nucleophilic ¬ CH2OH group to a highly nucleophilic alkoxide ion, ¬ CH2O: His 57 Asp 102 H2C CH2 O C – H O Ser 195 N CH2 N H His 57 Asp 102 CH2 O H2C O C O H Ser 195 N CH2 N H – O "Charge relay system" In the process, the anionic charge of Asp 102 was thought to be transferred, via a tautomeric shift of His 57, to Ser 195. The catalytic triad was therefore originally named the charge relay system. It is now realized, however, that such a mechanism is implausible because an alkoxide ion (pK 15) has far greater proton affinity than does His 57 (pK L 7, as measured by NMR techniques). How, then, can Asp 102 nucleophilically activate Ser 195? A possible solution to this conundrum has been pointed out by W.W. Cleland and Maurice Kreevoy and, independently, by John Gerlt and Paul Gassman. Proton transfers between hydrogen bonded groups (D¬ H p A) only occur at physiologically reasonable rates when the pK of the proton donor is no more than 2 or 3 pH units greater than that of the protonated form of the proton acceptor (the height of the kinetic barrier, G ‡, for the protonation of an acceptor by a more basic donor increases with the dif- ference between the pK’s of the donor and acceptor). However, when the pK’s of the hydrogen bonding donor (D) and acceptor (A) groups are nearly equal, the distinction between them breaks down: The hydrogen atom becomes more or less equally shared between them (D p H p A). Such low-barrier hydrogen bonds (LBHBs) are unusually short and strong (they are also known as short, strong hydrogen bonds): They have, as studies of model compounds in the gas phase indicate, association free energies as high as 40 to 80 kJ mol1 versus the 12 to 30 kJ mol1 for normal hydrogen bonds (the energy of the normally covalent D¬ H bond is subsumed into the low-barrier hydrogen bonding system) and a D p A length of 2.55 Å for O¬ H p O and 2.65 Å for N¬ H p O versus 2.8 to 3.1 Å for normal hydrogen bonds. LBHBs are unlikely to exist in dilute aqueous solution because water molecules, which are excellent hydrogen bonding donors and acceptors, effectively compete with D¬ H and A for hydrogen bonding sites. However, LBHBs may exist in nonaqueous solution and in the active sites of enzymes that exclude bulk solvent water. If so, an effective enzymatic “strategy” would be to convert a weak hydrogen bond in the Michaelis complex to a strong hydrogen bond in the transition state, thereby facilitating proton transfer while applying the difference in the free energy between the normal and low-barrier hydrogen bonds to preferentially binding the transition state. In fact, as Perry Frey has shown, the NMR spectrum of the proton linking His 57 to Asp 102 in chymotrypsin (which exhibits a particularly large downfield chemical shift indicative of deshielding) is consistent with the formation of an LBHB in the transition state (see Fig. 15-25b; the pK’s of protonated His 57 and Asp 102 are nearly equal in the anhydrous environment of the active site complex). This presumably promotes proton transfer from Ser 195 to His 57 as in the charge relay mechanism. Moreover, an ultrahigh (0.78 Å) resolution X-ray structure of Bacillus lentus subtilisin by Richard Bott reveals that the hydrogen bond between His 64 and Asp 32 of its catalytic triad has an unusually short N p O distance of 2.62 0.01 Å and that its H atom is nearly centered between the N and O atoms (note that this highly accurate protein X-ray structure is one of the very few in which H atoms are observed and in which short D p A distances are confidently measured). Although several studies, such as the foregoing, have revealed the existence of unusually short hydrogen bonds in enzyme active sites, it is far more difficult to demonstrate experimentally that they are unusually strong, as LBHBs are predicted to be. In fact, several studies of the strengths of unusually short hydrogen bonds in organic model compounds in nonaqueous solutions suggest that these hydrogen bonds are not unusually strong. Consequently, a lively debate has ensued as to the catalytic significance of LBHBs. Yet if enzymes do not form LBHBs, it remains to be explained how, in numerous widely accepted enzymatic mechanisms that we shall encounter, the conjugate base of an acidic group can abstract a proton from a far more basic group. 7884d_c15.qxd 1/23/03 12:28 PM Page 527 mac18 mac18:df_169:7884D: Section 15–3. Serine Proteases e. Much of a Serine Protease’s Catalytic Activity Arises from Preferential Transition State Binding Despite the foregoing, blocking the action of the catalytic triad through the specific methylation of His 57 by treating chymotrypsin with methyl-p-nitrobenzene sulfonate His 57 CH2 O H N1 + 3 O2N N S O CH3 O Methyl-p-nitrobenzene sulfonate His 57 CH2 H N1 + 3 O2N SO3– N+ CH3 yields an enzyme that is a reasonably good catalyst: It enhances the rate of proteolysis by as much as a factor of 2 106 over the uncatalyzed reaction, whereas the native enzyme has a rate enhancement factor of 1010. Similarly, the mutation of Ser 195, His 57, or even all three residues of the catalytic triad yields enzymes that enhance proteolysis rates by 5 104-fold over that of the uncatalyzed reaction. Evidently, the catalytic triad provides a nucleophile and is an alternate source and sink of protons (general acid– base catalysis). However, a large portion of chymotrypsin’s rate enhancement must be attributed to its preferential binding of the catalyzed reaction’s transition state. E. Zymogens Most proteolytic enzymes are biosynthesized as somewhat larger inactive precursors known as zymogens (enzyme precursors, in general, are known as proenzymes). In the case of digestive enzymes, the reason for this is clear: If these enzymes were synthesized in their active forms, they would digest the tissues that synthesized them. Indeed, acute pancreatitis, a painful and sometimes fatal condition that can be precipitated by pancreatic trauma, is characterized by the premature activation of the digestive enzymes synthesized by this gland. a. Serine Proteases Are Autocatalytically Activated Trypsin, chymotrypsin, and elastase are activated according to the following pathways: Trypsin. The activation of trypsinogen, the zymogen of trypsin, occurs as a two-stage process when trypsinogen enters the duodenum from the pancreas. Enteropeptidase, a single-pass transmembrane serine protease that is located 527 in the duodenal mucosa, specifically hydrolyzes trypsinogen’s Lys 15 ¬ Ile 16 peptide bond, thereby excising its Nterminal hexapeptide (Fig. 15-27). This yields the active enzyme, which has Ile 16 at its N-terminus. Since this activating cleavage occurs at a trypsin-sensitive site (recall that trypsin cleaves after Arg and Lys residues), the small amount of trypsin produced by enteropeptidase also catalyzes activation, generating more trypsin, etc.; that is, trypsinogen activation is autocatalytic. Chymotrypsin. Chymotrypsinogen is activated by the specific tryptic cleavage of its Arg 15 ¬ Ile 16 peptide bond to form -chymotrypsin (Fig. 15-28). -Chymotrypsin subsequently undergoes autolysis (self-digestion) to specifically excise two dipeptides, Ser 14–Arg 15 and Thr 147– Asn 148, thereby yielding the equally active enzyme -chymotrypsin (heretofore and hereafter referred to as chymotrypsin). The biochemical significance of this latter process, if any, is unknown. Elastase. Proelastase, the zymogen of elastase, is activated similarly to trypsinogen by a single tryptic cleavage that excises a short N-terminal polypeptide. b. Biochemical “Strategies” That Prevent Premature Zymogen Activation Trypsin activates pancreatic procarboxypeptidases A and B and prophospholipase A2 (the action of phospholipase A2 is outlined in Section 25-1) as well as the pancreatic serine proteases. Premature trypsin activation can consequently trigger a series of events that lead to pancreatic self-digestion. Nature has therefore evolved an elaborate defense against such inappropriate trypsin activation. We have already seen (Section 15-3D) that pancreatic trypsin inhibitor binds essentially irreversibly to any trypsin formed in the pancreas so as to inactivate it. Furthermore, the trypsin-catalyzed activation of trypsinogen (Fig. 15-27) occurs quite slowly, presumably because the unusually large negative charge of its highly evolutionarily conserved N-terminal hexapeptide repels the Asp at the back of trypsin’s specificity pocket. Finally, pancreatic zymogens are stored in intracellular vesicles called + H3N 10 Val (Asp)4 15 16 Lys Ile Val ... Trypsinogen enteropeptidase or trypsin + H3N Val (Asp)4 Lys + Ile Val ... Trypsin Activation of trypsinogen to form trypsin. Proteolytic excision of the N-terminal hexapeptide is catalyzed by either enteropeptidase or trypsin. The chymotrypsinogen residue numbering is used here; that is, Val 10 is actually trypsinogen’s N-terminus and Ile 16 is trypsin’s N-terminus. FIGURE 15-27 7884d_c15.qxd 528 1/23/03 12:28 PM Page 528 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis Activation of chymotrypsinogen by proteolytic cleavage. Both - and -chymotrypsin are See Kinemage enzymatically active. FIGURE 15-28 Chymotrypsinogen (inactive) 1 122 Cys Exercise 10-4 S Cys S 136 201 S Cys S 245 Cys trypsin Arg π-Chymotrypsin (active) 1 15 Ile 16 122 136 201 S S 245 S S chymotrypsin Ser Arg 14 Tyr Leu α-Chymotrypsin (active) 1 c. Zymogens Have Distorted Active Sites Since the zymogens of trypsin, chymotrypsin, and elastase have all their catalytic residues, why aren’t they enzymatically active? Comparisons of the X-ray structures of trypsinogen with that of trypsin and of chymotrypsinogen with that of chymotrypsin show that on activation, the newly liberated N-terminal Ile 16 residue moves from the surface of the protein to an internal position, where its free cationic amino group forms an ion pair with the invariant anionic Asp 194 (Fig. 15-21). Aside from this change, however, the structures of these zymogens closely resemble those of their corresponding active enzymes. Surprisingly, this resemblance includes their catalytic triads, an observation which led to the discovery that these zymogens are actually enzymatically active, albeit at a very low level. Careful comparisons of the corresponding enzyme and zymogen structures, however, revealed the reason for this low activity: The zymogens’ specificity pockets and oxyanion holes are improperly formed such that, for example, the amide NH of chymotrypsin’s Gly 193 points in the wrong direction to form a hydrogen bond with the tetrahedral intermediate (see Fig. 15-25). Hence, the zymogens’ very low enzymatic activity arises from their reduced ability to bind substrate productively and to stabilize the tetrahedral intermediate. These observations provide further structural evidence favoring the role of preferred transition state binding in the catalytic mechanism of serine proteases. 4 DRUG DESIGN The improvements in medical care over the past several decades are, in large measure, attributable to the development of a huge variety of drugs, which have eliminated + 147 148 Ala Ile 13 16 122 136 146 149 S S zymogen granules whose membranous walls are thought to be resistant to enzymatic degradation. 15 Thr Asn 201 245 S S or greatly relieved numerous human ailments. Such medications include antibiotics (which have enormously reduced the impact of infectious diseases), anti-inflammatory agents (which reduce the effects of inflammatory diseases such as arthritis), analgesics and anesthetics (which make modern surgical techniques possible), agents that reduce the incidence and severity of cardiovascular disease and stroke, antidepressants, antipsychotics, agents that inhibit stomach acid secretion (which prevent stomach ulcers and heartburn), agents to combat allergies and asthma, immunosuppressants (which make organ transplants possible), agents used for cancer chemotherapy, and a great variety of other substances. Early human cultures almost certainly recognized both the beneficial and toxic effects of indigenous plant and animal products and used many of them as “medications.” Unfortunately, most of these substances were useless or even harmful. Although there were sporadic attempts over the 2500 years preceding the modern era to formulate rational systems of drug discovery, they had little success because they were based mainly on unfounded theories and superstition (e.g., the doctrine of signatures stated that if a plant resembles a particular body part, it must be designed by nature to influence that body part) rather than observation and experiment. Consequently, at the beginning of the 20th century, only three known drugs, apart from folk medicines, were effective in treating specific diseases: (1) Digitalis, a heart stimulant extracted from the foxglove plant (Section 20-3A), was used to treat various heart conditions; (2) quinine (Section 26-4A), obtained from the bark and roots of the Cinchona tree, was used to treat malaria; and (3) mercury was used to treat syphilis (a cure that was often worse than the disease). It was not until several decades later that the rise of the scientific method coupled to the rapidly increasing knowledge of physiology, biochemistry, and chemistry led to effective methods of drug discovery. In fact, the vast majority of 7884d_c15.qxd 2/11/03 2:25 PM Page 529 ymac5:1st_ss_649: Section 15–4. Drug Design drugs in use today were discovered and developed in the past three decades. In this section we discuss the elements of drug discovery and pharmacology (the science of drugs, including their composition, uses, and effects). The section ends with a consideration of one of the major successes of modern drug discovery methods, HIV protease inhibitors. A. Techniques of Drug Discovery Most drugs act by modifying the function of a particular receptor in the body or in an invading pathogen. In most cases, the receptor is a protein to which the drug specifically binds. It may be an enzyme, a transmembrane channel that transports a specific substance into or out of a cell (Chapter 20), and/or a protein that participates in an interor intracellular signaling pathway (Chapter 19). In all of these cases, a substance that in binding to a receptor modulates its function is known as an agonist, whereas a substance that binds to a receptor without affecting its function but blocks the binding of agonists is called an antagonist. The biochemical and physiological effects of a drug and its mechanism of action are referred to as its pharmacodynamics. a. Drug Discovery Is a Complex Procedure How are new drugs discovered? Nearly all drugs that have been in use for over a decade were discovered by screening large numbers of synthetic compounds and natural products for the desired effect. Drug candidates that are natural products are usually discovered by the fractionation of the organisms in which they occur, which are often plants used in folk remedies of the conditions of interest. Humans having the condition whose treatment is being sought cannot be used as “guinea pigs” in this initial screening process, and even guinea pigs or other laboratory animals such as mice or dogs (if they can be made to be suitable models of the condition under consideration) are too expensive to use on the many thousands of compounds that are usually tested. Thus, in vitro screens are initially used, such as the degree of binding of a drug candidate to an enzyme that is implicated in a disease of interest, toxicity toward the target bacteria in the search for a new antibiotic, or effects on a line of cultured mammalian cells. However, as the number of drug candidates is winnowed down, more sensitive screens such as testing in animals are employed. A drug candidate that exhibits a desired effect is called a lead compound (or, colloquially, a lead). A good lead compound binds to its target receptor with a dissociation constant, KD 1 M. Such a high affinity is necessary to minimize a drug’s less specific binding to other macromolecules in the body and to ensure that only low doses of the drug need be taken. For enzyme inhibitors, the dissociation constant is the inhibitor’s KI or K¿I (Section 14-3). Other common measures of the effect of a drug are the IC50, the inhibitor concentration at which an enzyme exhibits 50% of its maximal activity; the ED50, the effective dose of a drug required to produce a therapeutic effect in 50% of a test sample; the TD50, the mean toxic dose 529 required to produce a particular toxic effect in animals; and the LD50, the mean lethal dose required to kill 50% of a test sample. For an inhibitor of an enzyme that follows MichaelisMenten kinetics, the IC50 is determined by measuring the ratio I /o for several values of [I] at constant [S], where I is the initial velocity of the enzyme when the inhibitor concentration is [I]. By dividing equation [14.24] by equation [14.38] with defined according to equation [14.37], we see that KM 3 S4 vI v0 KM 3S4 KM 3 S4 3I4 KM a1 b 3S4 K1 When I /o 0.5 (50% inhibition), 3I4 3 IC50 4 KI a1 3S 4 KM b [15.13] [15.14] Consequently, if the measurements of I /o are made with [S] KM, then [IC50] KI. The ratio TD50ED50 is defined as a drug’s therapeutic index, the ratio of the dose of the drug that produces toxicity to that which produces the desired effect. It is, of course, preferable that a drug have a high therapeutic index, but this is not always possible. b. Cathepsin K Is a Drug Target for Osteoporosis The development of genomic sequencing techniques (Section 7-2B) and hence the characterization of tens of thousands of previously unknown genes is providing an enormous number of potential drug targets. For example, osteoporosis, a condition that afflicts postmenopausal women and elderly men, is characterized by the progressive loss of bone mass leading to a greatly increased frequency of bone fracture, particularly of the hip, spine, and wrist. Bones consist of a protein matrix that is 90% type I collagen (Section 8-2B), in which spindle- or plate-shaped crystals of hydroxyapatite, Ca5(PO4)3OH, are embedded. Bones are by no means static structures. They undergo continuous remodeling through the countervailing action of two types of bone cells: osteoblasts, which synthesize bone’s organic matrix in which its mineral component is laid down; and osteoclasts, which solubilize mineralized bone matrix through the secretion of proteolytic enzymes into an extracellular bone resorption pit, which is maintained at pH 4.5. The acidic solution dissolves the bone’s mineral component, thereby exposing its protein matrix to proteolytic degradation. Osteoporosis arises when bone resorption outstrips bone formation. In the search for a drug target for osteoporosis, a cDNA library (Sections 5-5E and 5-5F) was prepared from an osteoclastoma (a cancer derived from osteoclasts; normally osteoclasts are very rare cells). Around 4% of these cDNAs encode a heretofore unknown protease, which was named cathepsin K (cathepsins are proteases that occur in the lysosome). Further studies, both at the cDNA and protein levels, indicated that cathepsin K is only expressed at high levels in osteoclasts. Microscopic examination of osteoclasts that had been stained with antibodies directed against 7884d_c15.qxd 530 1/29/03 12:01 PM Page 530 mac46 mac46:1352_mjw:7884d: Chapter 15. Enzymatic Catalysis cathepsin K revealed that this enzyme is localized at the contact site between osteoclasts and the bone resorption pit. Subsequently, it was shown that mutations in the gene encoding cathepsin K are the cause of pycnodysostosis, a rare hereditary disease which is characterized by hardened and fragile bones, short stature, skull deformities, and osteoclasts that demineralize bone normally but do not degrade its protein matrix. Evidently, cathepsin K functions to degrade the protein matrix of bone and hence is an attractive drug target for the treatment of osteoporosis. c. SARs and QSARs Are Useful Tools for Drug Discovery A lead compound is used as a point of departure to design more efficacious compounds. Experience has shown that even minor modifications to a drug candidate can result in major changes in its pharmacological properties. Thus, one might place methyl, chloro, hydroxyl, or benzyl groups at various places on a lead compound in an effort to improve its pharmacodynamics. For most drugs in use today, 5 to 10 thousand related compounds were typically synthesized in generating the medicinally useful drug. These were not random procedures but were guided by experience as medicinal chemists tested various derivatives of a lead compound: For those compounds that had improved efficacy, derivatives were made and tested; etc. This process has been systematized through the use of structure–activity relationships (SARs): the determination, via synthesis and screening, of which groups on a lead compound are important for its drug function and which are not. For example, if a phenyl group on a lead compound interacts hydrophobically with a flat region of its receptor, then hydrogenating the phenyl ring to form a nonplanar cyclohexane ring will yield a compound with reduced affinity for the receptor. A logical extension of the SAR concept is to quantify it, that is, to determine a quantitative structure–activity relationship (QSAR). This idea is based on the premise that there is a relatively simple mathematical relationship between the biological activity of a drug and its physicochemical properties. For instance, if the hydrophobicity of a drug is important for its biological activity, then changing the substituents on the drug so as to alter its hydrophobicity will affect its activity. A measure of the substance’s hydrophobicity is its partition coefficient, P, between the two immiscible solvents, octanol and water, at equilibrium: P concentration of drug in octanol concentration of drug in water [15.15] Biological activity may be expressed as 1C, where C is the drug concentration required to achieve a specified level of biological function (e.g., IC50). Then a plot of log 1C versus log P (the use of logarithms keeps the plot on a manageable scale) for a series of derivatives of the lead compound having a relatively small range of log P values often indicates a linear relationship (Fig. 15-29a), which can therefore be expressed: (a) 1 log __ C 1 log a b k1 log P k2 C log P (b) Here k1 and k2 are constants, whose optimum values in this QSAR can be determined by computerized curve-fitting methods. For compounds with a larger range of log P values, it is likely that a plot of log 1C versus log P will have a maximum value (Fig. 15-29b) and hence be better described by a quadratic equation: 1 log a b k1 1log P2 2 k2 log P k3 C 1 log __ C log P Hypothetical QSAR plots of log(1/C) versus log P for a series of related compounds. (a) A plot that is best described by a linear equation. (b) A plot that is best described by a quadratic equation. [15.16] [15.17] Of course, the biological activities of few substances depend only on their hydrophobicities. A QSAR can therefore simultaneously take into account several physicochemical properties of substituents such as their pK values, van der Waals radii, hydrogen bonding energy, and conformation. The values of the constants for each of the terms in a QSAR is indicative of the contribution of that term to the drug’s activity. The use of QSARs to optimize the biological activity of a lead compound has proven to be a valuable tool in drug discovery. FIGURE 15-29 d. Structure-Based Drug Design Since the mid 1980s, dramatic advances in the speed and precision with which a macromolecular structure can be 7884d_c15.qxd 1/23/03 12:28 PM Page 531 mac18 mac18:df_169:7884D: Section 15–4. Drug Design determined by X-ray crystallography and NMR (Section 8-3A) have enabled structure-based drug design, a process that greatly reduces the number of compounds that need be synthesized in a drug discovery program. As its name implies, structure-based drug design (also called rational drug design) uses the structure of a receptor in complex with a drug candidate to guide the development of more efficacious compounds. Such a structure will reveal, for example, the positions of the hydrogen bonding donors and acceptors in a receptor binding site as well as cavities in the binding site into which substituents might be placed on a drug candidate to increase its binding affinity for the receptor. These direct visualization techniques are usually supplemented with molecular modeling tools such as the computation of the minimum energy conformation of a proposed derivative, quantum mechanical calculations that determine its charge distribution and hence how it would interact electrostatically with the receptor, and docking simulations in which an inhibitor candidate is computationally modeled into the binding site on the receptor to assess potential interactions. Structure-based drug design is an iterative process: The structure of the receptor in complex with a compound with improved properties is determined in an effort to further improve its properties. e. Combinatorial Chemistry and High-Throughput Screening As structure-based methods were developed, it appeared that they would become the dominant mode of drug discovery. However, the recent advent of combinatorial chemistry techniques to rapidly and inexpensively synthesize large numbers of related compounds combined with the development of robotic high-throughput screening techniques has caused the drug discovery “pendulum” to again swing toward the “make-many-compounds-and-seewhat-they-do” approach. A familiar example of combinatorial chemistry is the parallel synthesis of the large number of different oligonucleotides on a DNA chip (Section 7-6B). Similarly, if a lead compound can be synthesized in a stepwise manner from several smaller modules, then the substituents on each of these modules can be varied in parallel to produce a library of related compounds (e.g., Fig. 15-30). A variety of synthetic techniques have been developed that permit the combinatorial synthesis of thousands of related compounds in a single procedure. Thus, whereas investigations into the importance of a hydrophobic group at a particular position in a lead compound might previously have prompted the individual syntheses of only the ethyl, propyl, and benzyl derivatives of the compound, the use of combinatorial synthesis would permit the generation of perhaps 100 different groups at that position. This R2CHO N O R1 O O O B. Introduction to Pharmacology The in vitro development of an effective drug candidate is only the first step in the drug development process. Besides causing the desired response in its isolated target receptor, a useful drug must be delivered in sufficiently high concentration to this receptor where it resides in the human body without causing unacceptable side effects. a. Pharmacokinetics Is a Multifaceted Phenomenon The most convenient form of drug administration is orally (by mouth). In order to reach its target receptor, a drug administered in this way must surmount a series of formidable barriers: (1) It must be chemically stable in the highly acidic (pH 1) environment of the stomach and must not be degraded by the digestive enzymes in the gastrointestinal tract; (2) it must be absorbed from the gastrointestinal tract into the bloodstream, that is, it must pass through several cell membranes; (3) it must not bind too tightly to other substances in the body (e.g., lipophilic substances tend to be absorbed by certain plasma proteins and by fat tissue; anions may be bound by plasma proteins, mainly albumin; and cations may be bound by nucleic acids); (4) it must survive derivatization by the battery of enzymes, mainly in the liver, that function to detoxify xenobiotics (foreign compounds), as discussed below (note that the intestinal blood flow drains directly into the liver via the portal vein, so that the liver processes all orally ingested substances before they reach the rest of the body); (5) it must avoid rapid excretion by the kidneys; (6) it must pass from the capillaries to its target tissue; (7) if it is targeted to the brain, it must cross the blood–brain barrier, which blocks the passage of most polar substances; and (8) if it is targeted to an intracellular receptor, it must pass through the plasma membrane and, possibly, other intracellular membranes. The ways in which a drug interacts with these various barriers is known as its pharmacokinetics. Thus, the bioavailability of a drug (the extent to which it reaches its site of action, which is usually taken to be the systemic circulation) depends on both the dose given and its pharmacokinetics. Of course, barriers (1) and (2) can be circumvented by injecting the drug [e.g., some forms O R3NH2 N R1 would far more effectively map out the potential range of the substituent and possibly identify an unexpectedly active analog. Interestingly, QSAR and computational techniques have been combined in the development of “virtual combinatorial chemistry,” a procedure in which libraries of compounds are computationally “synthesized” and “analyzed” to predict their efficacy, thereby again reducing the number of compounds that must actually be synthesized in order to generate an effective drug. R2 R2 R1 531 H N N H R3 O FIGURE 15-30 The combinatorial synthesis of arylidene diamides. If ten different variants of each R group are used in the synthesis, then 1000 different derivatives will be synthesized. 7884d_c15.qxd 532 2/10/03 9:25 AM Page 532 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis of penicillin (Fig. 11-25) must be injected because their functionally essential -lactam rings are highly susceptible to acid hydrolysis], but this mode of drug delivery is undesirable for long-term use. Since the pharmacokinetics of a drug candidate is as important to its efficacy as is its pharmacodynamics, both must be optimized in producing a medicinally useful drug. The following empirically based rules, formulated by Christopher Lipinski and known as Lipinski’s “rule of five,” state that a compound is likely to exhibit poor absorption or permeation if: 1. Its molecular mass is greater than 500 D. 2. It has more than 5 hydrogen bond donors (expressed as the sum of its OH and NH groups). 3. It has more than 10 hydrogen bond acceptors (expressed as the sum of its N and O atoms). 4. Its value of log P is greater than 5. Drug candidates that disobey Rule 1 are likely to have low solubilities and to only pass through cell membranes with difficulty; those that disobey Rules 2 and/or 3 are likely to be too polar to pass through cell membranes; and those that disobey Rule 4 are likely to be poorly soluble in aqueous solution and hence unable to gain access to membrane surfaces. Thus, the most effective drugs are usually a compromise; they are neither too lipophilic nor too hydrophilic. In addition, their pK values are usually in the range 6 to 8 so that they can readily assume both their ionized and unionized forms at physiological pH’s. This permits them to cross cell membranes in their unionized form and to bind to their receptor in their ionized form. However, since the concentration of a drug at its receptor depends, as we saw, on many different factors, the pharmacokinetics of a drug candidate may be greatly affected by even small chemical changes. QSARs and other computational tools have been developed to predict these effects but they are, as yet, rather crude. b. Toxicity and Adverse Reactions Eliminate Most Drug Candidates The final criteria that a drug candidate must meet are that its use be safe and efficacious in humans. Tests for these properties are initially carried out in animals, but since humans and animals often react quite differently to a particular drug, the drug must ultimately be tested in humans through clinical trials. In the United States, clinical trials are monitored by the Food and Drug Administration (FDA) and have three increasingly detailed (and expensive) phases: Phase I. This phase is primarily designed to test the safety of a drug candidate but is also used to determine its dosage range and the optimal dosage method (e.g., orally vs injection) and frequency. It is usually carried out on a small number (20–100) of normal, healthy volunteers, but in the case of a drug candidate known to be highly toxic (e.g., a cancer chemotherapeutic agent), it is carried out on volunteer patients with the target disease. Phase II. This phase mainly tests the efficacy of the drug against the target disease in 100 to 500 volunteer patients but also refines the dosage range and checks for side effects. The effects of the drug candidate are usually assessed via single blind tests, procedures in which the patient is unaware of whether he/she has received the drug or a control substance. Usually the control substance is a placebo (an inert substance with the same physical appearance, taste, etc., as the drug being tested) but, in the case of a life-threatening disease, it is an ethical necessity that the control substance be the best available treatment against the disease. Phase III. This phase monitors adverse reactions from long-term use as well as confirming efficacy in 1000 to 5000 patients. It pits the drug candidate against control substances through the statistical analysis of carefully designed double blind tests, procedures in which neither the patients nor the clinical investigators evaluating the patients’ responses to the drug know whether a given patient has received the drug or a control substance. This is done to minimize bias in the subjective judgments the investigators must make. Currently, only about 5 drug candidates in 5000 that enter preclinical trials reach clinical trials. Of these, only one, on average, is ultimately approved for clinical use, with 40% of drug candidates passing Phase I trials and 50% of those passing Phase II trials (most drug candidates that enter Phase III trials are successful). In recent years, the preclinical portion of a drug discovery process has averaged 3 years to complete, whereas successful clinical trials have usually required an additional 7 to 10 years. These successive stages of the drug discovery process are increasingly expensive, so that to successfully bring a drug to market costs, on average, around $300 million. The most time-consuming and expensive aspect of a drug development program is identifying a drug candidate’s rare adverse reactions. Nevertheless, it is not an uncommon experience for a drug to be brought to market only to be withdrawn some months or years later when it is found to have caused unanticipated life-threatening side effects in as few as 1 in 10,000 individuals (the search for new applications of an approved drug and its postmarketing surveillance are known as its Phase IV clinical trials). For example, in 1997, the FDA withdrew its approval of the drug fenfluramine (fen), CH3 CH2 CH NH CH2 CH3 CH3 CH2 CF3 Fenfluramine C NH2 CH3 Phentermine which it had approved in 1973 for use as an appetite suppressant in short-term (a few weeks) weight loss programs. Fenfluramine had become widely prescribed, often for ex- 7884d_c15.qxd 1/29/03 12:01 PM Page 533 mac46 mac46:1352_mjw:7884d: Section 15–4. Drug Design 533 tended periods, together with another appetite suppressant, phentermine (phen; approved in 1959), a combination known as fen-phen (although the FDA had not approved of the use of the two drugs in combination, once it approves a drug for any purpose, a physician may prescribe it for any other purpose). The withdrawal of fenfluramine was prompted by over 100 reports of heart valve damage in individuals (mostly woman) who had taken fen-phen for an average of 12 months (phentermine was not withdrawn because the evidence indicated that fenfluramine was the responsible agent). This rare side effect had not been observed in the clinical trials of fenfluramine, in part because, being an extremely unusual type of drug reaction, it had not been screened for. c. The Cytochromes P450 Metabolize Most Drugs Why is it that a drug that is well tolerated by the majority of patients can pose such a danger to others? Differences in reactions to drugs arise from genetic differences among individuals as well as differences in their disease states, other drugs they are taking, age, sex, and environmental factors. The cytochromes P450, which function in large part to detoxify xenobiotics and participate in the metabolic clearance of the majority of drugs in use, provide instructive examples of these phenomena. The cytochromes P450 constitute a superfamily of heme-containing enzymes that occur in nearly all living organisms, from bacteria to mammals [their name arises from the characteristic 450-nm peak in their absorption spectra when reacted in their Fe(II) state with CO]. Humans express 100 isozymes (catalytically and structurally similar but genetically distinct enzymes from the same organism; also called isoforms) of cytochromes P450, mainly in the liver but also in other tissues (its various isozymes are named by the letters “CYP” followed by a number designating its family, an uppercase letter designating its subfamily, and often another number; e.g., CYP2D6). These monooxygenases (Fig. 15-31), which in animals are embedded in the endoplasmic reticulum membrane, catalyze reactions of the sort RH O2 2H 2e ∆ ROH H 2O The electrons (e) are supplied by NADPH, which passes them to cytochrome P450’s heme prosthetic group via the intermediacy of the enzyme cytochrome P450 reductase. Here RH represents a wide variety of usually lipophilic compounds for which the different cytochromes P450 are specific. They include polycyclic aromatic hydrocarbons [PAHs, frequently carcinogenic (cancer-causing) compounds that are present in tobacco smoke, broiled meats, and other pyrolysis products], polycyclic biphenyls (PCBs, which were widely used in electrical insulators and as plasticizers and are also carcinogenic), steroids (in whose syntheses cytochromes P450 participate; Sections 25-6A and 25-6C), and many different types of drugs. The xenobiotics are thereby converted to a more water-soluble form, which aids in their excretion by the kidneys. Moreover, the newly generated hydroxyl groups are often enzymatically conju- X-Ray structure of cytochrome P450CAM from Pseudomonas putida showing its active site region. The heme group, the Cys side chain that axially ligands its Fe atom, and the enzyme’s lipophilic substrate thiocamphor are shown in ball-and-stick form with N blue, O red, S yellow, Fe orange, and the C atoms of the heme, its liganding Cys side chain, and the thiocamphor green, cyan, and pale blue-green, respectively. The bonds liganding the Fe are gray. [Based on an X-ray structure by Thomas Poulos, University of California at Irvine. PDBid 8CPP.] FIGURE 15-31 gated (covalently linked) to polar substances such as glucuronic acid (Section 11-1C), glycine, sulfate, and acetate, which further enhances aqueous solubility. The many types of cytochromes P450 in animals, which have different substrate specificities (although these specificities tend to be broad and hence often overlap), are thought to have arisen in response to the numerous toxins which plants produce, presumably to discourage animals from eating them. Drug–drug interactions are often mediated by cytochromes P450. For example, if drug A is metabolized by or otherwise inhibits a cytochrome P450 isozyme that metabolizes drug B, then coadministering drugs A and B will cause the bioavailability of drug B to increase above the value it would have had if it alone had been administered. This phenomenon is of particular concern if drug B has a low therapeutic index. Conversely, if, as is often the case, drug A induces the increased expression of the cytochrome P450 isozyme that metabolizes it and drug B, then coadministering drugs A and B will reduce drug B’s bioavailability, a phenomenon that was first noted when certain antibiotics caused oral contraceptives to lose their efficacy. Moreover, if drug B is metabolized to a toxic product, its increased rate of reaction may result in an adverse reaction. Environmental pollutants such as PAHs or PCBs are also known to induce the expression of specific cytochrome P450 isozymes and thereby alter the rates at which certain drugs are metabolized. Finally, some of these same effects may occur in patients with liver disease, as well as arising from age-based, gender-based, and individual differences in liver physiology. Although the cytochromes P450 presumably evolved to detoxify and/or help eliminate harmful substances, in sev- 7884d_c15.qxd 534 1/23/03 12:28 PM Page 534 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis O O HO C H N C CH3 C CH3 N cytochrome P450 O2 N The metabolic reactions of acetaminophen that convert it to its conjugate with glutathione. FIGURE 15-32 O CH3 spontaneous H2O H2O OH O OH Acetaminophen (N-acetyl-p-aminophenol) Acetimidoquinone SH COO– + H3N CH CH2 O O CH2 CH2 C NH CH C NH CH2 COO– CH2 COO– Glutathione (-L-Glutamyl-L-cysteinyl-glycine) O C H CH3 N S COO– + H3N CH CH2 O CH2 C OH NH CH2 O CH C NH Acetaminophen–glutathione conjugate eral cases they have been shown to participate in converting relatively innocuous compounds to toxic agents. For example, acetaminophen (Fig. 15-32), a widely used analgesic and antipyretic (fever reducer), is quite safe when taken in therapeutic doses (1.2 g/day for an adult) but in large doses (10 g) is highly toxic. This is because, in therapeutic amounts, 95% of the acetaminophen present is enzymatically glucuronidated or sulfated at its ¬ OH group to the corresponding conjugates, which are readily excreted. The remaining 5% is converted, through the action of a cytochrome P450 (CYP2E1), to acetimidoquinone (Fig. 15-32), which is then conjugated with glutathione, a tripeptide with an unusual -amide bond that participates in a wide variety of metabolic processes (Section 26-4C). However, when acetaminophen is taken in large amounts, the glucuronidation and sulfation pathways become saturated and hence the cytochrome P450-mediated pathway becomes increasingly important. If hepatic (liver) glutathione is depleted faster than it can be replaced, acetimidoquinone, a reactive compound, instead conjugates with the sulfhydryl groups of cellular proteins, resulting in often fatal hepatotoxicity. Many of the cytochromes P450 in humans are unusually polymorphic, that is, there are several common alleles (variants) of the genes encoding these enzymes. Alleles that cause diminished, enhanced, and qualitatively altered rates of drug metabolism have been characterized for many of the cytochromes P450. The distributions of these various alleles differ markedly among ethnic groups and hence probably arose to permit each group to cope with the toxins in its particular diet. Polymorphism in a given cytochrome P450 results in differences between individuals in the rates at which they metabolize certain drugs. For instance, in cases that a cytochrome P450 variant has absent or diminished activity, otherwise standard doses of a drug that the enzyme normally metabolizes may cause the bioavailability of the drug to reach toxic levels. Conversely, if a particular P450 enzyme has enhanced activity (usually because the gene encoding it has been duplicated one or more times), higher than normal doses of a drug that the enzyme metabolizes would have to be administered to obtain the required therapeutic effect. However, if the drug is metabolized to a toxic product, this may result in an adverse reaction. Several known P450 variants have altered substrate specificities and hence produce unusual metabolites, which also may cause harmful side effects. Experience has amply demonstrated that there is no such thing as a drug that is entirely free of adverse reactions. However, as the enzymes and their variants that participate in drug metabolism are characterized and rapid and inexpensive genotyping methods are developed, it may be- 7884d_c15.qxd 1/23/03 12:28 PM Page 535 mac18 mac18:df_169:7884D: Section 15–4. Drug Design come possible to tailor drug treatment to an individual’s genetic makeup rather than to the population as a whole. C. HIV Protease and Its Inhibitors Acquired immunodeficiency syndrome (AIDS), the only major epidemic attributable to a previously unknown pathogen to appear in the 20th century (it was first described in 1981), is caused by human immunodeficiency virus type 1 (HIV-1; the closely related HIV-2, which we shall not explicitly discuss here, also causes AIDS and has a similar response to drugs). HIV-1, which was discovered in 1983, is a retrovirus, a family of viruses that were independently characterized in 1970 by David Baltimore and Howard Temin. The retroviral genome is a single-stranded RNA that reproduces inside its host cell by transcribing the RNA to double-stranded DNA in a process mediated by the virally encoded enzyme reverse transcriptase (Section 30-4C). The DNA is then inserted into the host cell’s chromosomal DNA by a viral enzyme named integrase and is passively replicated along with the cell’s DNA. However, under activating conditions (which for HIV-1 often is an infection by another pathogen), the retroviral DNA is transcribed, the proteins it encodes are expressed and inserted in or anchored to the host cell plasma membrane, and new virions (virus particles) are produced by the budding out of a viral protein-laden segment of plasma membrane so as to enclose viral RNA (Fig. 15-33). HIV-1 is targeted to and specifically replicates within helper T cells, essential components of the immune system (Section 35-2A). Unlike most types of retroviruses, HIV-1 eventually kills the cells producing it. Although the helper T cells within which HIV-1 are actively replicating are often destroyed by the immune system, those within which the HIV-1 is latent (its DNA is not being transcribed) are not detected by the immune system and hence provide a reservoir of HIV-1 (other types of cells also harbor HIV-1). Consequently, over a several year period after the initial 535 infection (during most of which the host exhibits no obvious symptoms), the host’s immune system is steadily depleted until it has deteriorated to the point that the host regularly falls victim to and is eventually killed by opportunistic pathogens that individuals with normally functioning immune systems can readily withstand. It is this latter stage of an HIV infection that is called AIDS. In the absence of effective therapy, AIDS is almost invariably fatal. Through the year 2002, an estimated 30 million people had died of AIDS and an estimated 42 million others, largely in sub-Saharan Africa, were HIV-positive, numbers that are increasing at the rate of 5 million per year. As a consequence of this global catastrophe, HIV has been characterized and effective countermeasures against it have been devised faster than for any other pathogen in history. a. Reverse Transcriptase Inhibitors Are Only Partially Effective The first drug to be approved by the FDA (in 1987) to fight AIDS was 3-azido-3-deoxythymidine (AZT; zidovudine), HOCH2 H _ N T O H H H + N N H 3-Azido-3-deoxythymidine (AZT; zidovudine) which had first been synthesized in 1964 as a possible anticancer agent (it was ineffective). AZT is a nucleoside analog that, on enzymatic conversion to its triphosphate in the cell (the plasma membrane is impermeable to nucleoside triphosphates), inhibits HIV-1 reverse transcriptase, as do the several other drugs (Section 30-4C) that the FDA had approved to treat AIDS prior to 1996. Unfortunately, these agents only slow the progression of an HIV infection but do not stop it. This is in part because they are toxic, mainly FIGURE 15-33 The assembly, budding, and maturation of HIV-1. SU is the surface glycoprotein gp120 and TM is the transmembrane protein gp41. [After Turner, B.G. and Summers, M.F., J. Mol. Biol. 285, 4 (1999).] 7884d_c15.qxd 536 1/23/03 12:28 PM Page 536 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis (a) (a) I MA II III CA p1 IV NC p6 gag I MA II III CA p1 V TF VI VII PR RT VIII RN IN gag–pol (b) Cleavage site HIV-1 polyproteins. (a) The organization of the HIV-1 gag and gag–pol polyproteins. The symbols used are MA, matrix protein; CA, capsid protein; NC, nucleocapsid protein; TF, transmembrane protein; PR, protease; RT, reverse transcriptase; RN, ribonuclease; and IN, integrase. (b) The sequences flanking the HIV-1 protease cleavage sites (red bonds) indicated in Part a. FIGURE 15-34 to the bone marrow cells that are blood cell precursors, and hence cannot be taken in large doses. More important, however, is that reverse transcriptase, unlike most other DNA polymerases (Section 30-2A), cannot correct its mistakes and hence frequently generates mutations (about one per 104 bp and, since the viral genome consists of 104 bp, each viral genome bears, on average, one new mutation). Consequently, under the selective pressure of an anti-HIV drug such as AZT, the drug’s target receptor rapidly evolves to a drug-resistant form. b. HIV-1 Polyproteins Are Cleaved by HIV-1 Protease HIV-1, as do other retroviruses, synthesizes its proteins in the form of polyproteins, which each consist of several tandemly linked proteins (Fig. 15-34). HIV-1 encodes two polyproteins, gag (55 kD) and gag–pol (160 kD), which are both anchored to the plasma membrane via N-terminal myristoylation (Section 12-3B). These polyproteins are then cleaved to their component proteins through the action of HIV-1 protease, but only after this enzyme has excised itself from gag–pol. This process occurs only after the virion has budded off from the host cell and results in a large structural reorganization of the virion (Fig. 15-33). The virion is thereby converted from its noninfectious immature form to its pathogenic mature form. If HIV-1 protease is inactivated, either mutagenically or by an inhibitor, the virion remains noninfectious. Hence HIV-1 protease is an opportune drug target. c. Aspartic Proteases and Their Catalytic Mechanism HIV-1 protease is a member of the aspartic protease family (also known as acid proteases), so called because I II III IV V VI VII VIII Sequence ... Ser -Gln-Asn- Tyr — Pro - Ile - Val - Gln ... ... Ala -Arg- Val -Leu — Ala -Glu- Ala -Met ... ... Ala - Thr - Ile -Met — Met-Gln-Arg- Gly ... ... Pro - Gly -Asn- Phe — Leu-Gln- Ser -Arg ... ... Ser -Phe-Asn- Phe — Pro -Gln- Ile - Thr ... ... Thr -Leu-Asn- Phe — Pro - Ile - Ser - Pro ... ... Ala -Glu- Thr - Phe — Tyr - Val -Asp- Gly ... ... Arg- Lys - Ile -Leu — Phe -Leu-Asp- Gly ... these enzymes all contain catalytically essential Asp residues that occur in the signature sequence Asp– Thr/Ser–Gly. Humans have several known aspartic proteases including pepsin, a digestive enzyme secreted by the stomach (its specificity is indicated in Table 7-2) that functions at pH 1 and which was the first enzyme to be recognized (named in 1825 by T. Schwann); chymosin (formerly rennin), a stomach enzyme, occurring mainly in infants, that specifically cleaves a Phe–Met peptide bond in the milk protein -casein, thereby causing milk to curdle, making it easier to digest (calf stomach chymosin has been used for millennia to make cheese); cathepsins D and E, lysosomal proteases that function to degrade cellular proteins; renin, which participates in the regulation of blood pressure and electrolyte balance (Fig. 15-35); and -secretase (also known as memapsin 2), a transmembrane protein common in brain that participates in cleaving A precursor protein to yield amyloid- protein (A), which is implicated in Alzheimer’s disease (Section 9-5B). In addition, many fungi secrete aspartic proteases, presumably to aid them in invading the tissues they colonize. Eukaryotic aspartic proteases are 330-residue monomeric proteins. The X-ray structure of pepsin (Fig. 15-36a), which closely resembles those of other eukaryotic aspartic proteases, reveals that this croissant-shaped protein consists of two homologous domains that are related by approximate 2-fold symmetry (although only about 25 residues in the core sheets of each domain are closely related by this symmetry). Each domain contains a catalytically essential Asp in an analogous position. The Xray structures of enzyme–inhibitor complexes of various aspartate proteases indicate that substrates bind in a prominent cleft between the two domains that could ac- 7884d_c15.qxd 3/24/03 1:01 PM Page 537 mac85 Mac 85:1st shift: 1268_tm:7884d: Section 15–4. Drug Design 1 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His13 Angiotensinogen H2O renin 1 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu10 + Val-Ile-His Angiotensin I H2O angiotensin converting enzyme (ACE) 1 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe8 + His-Leu Angiotensin II Renin participation in blood pressure regulation. Renin proteolytically cleaves the 13-residue polypeptide angiotensinogen to the 10-residue polypeptide angiotensin I. This latter peptide is then cleaved by angiotensin converting enzyme (ACE) to the 8-residue polypeptide angiotensin II, which, on binding to its receptor, induces vasoconstriction and retention of Na and water by the kidneys, resulting in increased blood pressure. Consequently there have been considerable efforts to develop both renin and ACE inhibitors for the control of hypertension (high blood pressure), although as yet, only ACE inhibitors have been approved as drugs. FIGURE 15-35 537 What is the catalytic mechanism of eukaryotic aspartic proteases? Proteolytic enzymes, in general, have three essential catalytic components: 1. A nucleophile to attack the carbonyl C atom of the scissile peptide to form a tetrahedral intermediate (Ser 195 serves this function in trypsin; Fig. 15-23). 2. An electrophile to stabilize the negative charge that develops on the carbonyl O atom of the tetrahedral intermediate (the H-bonding donors lining the oxyanion hole, Gly 193 and Ser 195, do so in trypsin; Fig. 15-25). 3. A proton donor so as to make the amide N atom of the scissile peptide a good leaving group (the imidazolium group of His 57 in trypsin; Fig. 15-23). commodate an 8-residue polypeptide segment in an extended sheetlike conformation. The active site Asp residues are located at the base of this cleft (Fig. 15-36a). Pepsin’s pH rate profile (Section 14-4) suggests that it has two ionizable essential residues, one with pK L 1.1 and the other with pK L 4.7, which are almost certainly the carboxyl groups of its essential Asp residues. At the pH of the stomach, the Asp residue with pK 4.7 is protonated and that with pK 1.1 is partially ionized. This suggests that the ionized carboxyl group acts as a nucleophile to form the putative tetrahedral intermediate. However, no covalent intermediate between an aspartic protease and its substrate has ever been detected. The two active site Asp residues in eukaryotic aspartic proteases are in close proximity and both appear to form hydrogen bonds to a bridging water molecule that is present in several X-ray structures of eukaryotic aspartic proteases (Fig. 15-36b). This, together with a variety of enzymological and kinetic data, led Thomas Meeks to propose (a) (b) FIGURE 15-36 X-Ray structure of pepsin. (a) Ribbon diagram in which the N-terminal domain (residues 1–172) is gold, the C-terminal domain (residues 173–326) is cyan, the side chains of the active site Asp residues are shown in ball-and-stick form with C green and O red, and the water molecule that is bound by these Asp side chains is represented by a large red sphere. The protein is viewed with the pseudo-2-fold axis relating core portions of the two domains tipped from vertical toward the viewer. (b) Enlarged view of the active site Asp residues and their bound water molecule indicating the lengths (in Å) of possible hydrogen bonds (thin gray bonds). The X-ray structures of other aspartic proteases exhibit similar interatomic distances. [Based on an X-ray structure by Anita Sielecki and Michael James, University of Alberta, Edmonton, Canada. PDBid 4PEP.] 7884d_c15.qxd 3/24/03 1:01 PM Page 538 mac85 Mac 85:1st shift: 1268_tm:7884d: Chapter 15. Enzymatic Catalysis 538 the following catalytic mechanism for aspartic proteases (Fig. 15-37): 1. An active site Asp carboxylate group, acting as a general base, activates the bound water molecule, the socalled lytic water, to nucleophilically attack the scissile peptide’s carbonyl C as an OH ion. Proton donation (general acid catalysis) by the second, previously uncharged active site Asp stabilizes the oxyanion that would otherwise form in the resulting tetrahedral intermediate. d. HIV-1 Protease Inhibitors Are Effective Anti-AIDS Agents HIV-1 protease differs from eukaryotic aspartic proteases in that it is a homodimer of 99-residue subunits. Nevertheless, its X-ray structure (Fig. 15-38a), determined independently in 1989 by Alexander Wlodawer, by Manual Navia and Paula Fitzgerald, and by Tom Blundell, closely resembles those of eukaryotic aspartic proteases. Thus, HIV-1 protease has the enzymatically unusual property that its single active site is formed by two identical sym- 2. The N atom of the scissile peptide is protonated by the first Asp (general acid catalysis) resulting, through charge rearrangement and proton transfer to the second Asp (general base catalysis), in amide bond scission. Aspartic proteases are inhibited by compounds with tetrahedral carbon atoms at a position mimicking a scissile peptide bond (see below). This strongly suggests that these enzymes preferentially bind their transition states (transition state stabilization), thereby enhancing catalysis. H H R N C R N R C O H O C R O 1 H O O –O H – C O C O Asp (a) H H O O H Asp C O O Asp Michaelis complex Asp Tetrahedral intermediate 2 R O R H C + O H N H (b) H O C –O O Asp C Asp Products Catalytic mechanism of aspartic proteases. (1) The nucleophilic attack of the enzyme-activated water molecule (red) on the carbonyl carbon atom of the scissile peptide bond (green) to form the tetrahedral intermediate. This reaction step is promoted by general base catalysis by the Asp on the right and general acid catalysis by the Asp on the left (blue). (2) The decomposition of the tetrahedral intermediate to form products via general acid catalysis by the Asp on the right and general base catalysis by the Asp on the left. FIGURE 15-37 X-Ray structure of HIV-1 protease. (a) Uncomplexed and (b) in complex with its inhibitor saquinavir (structural formula in Fig. 15-41). In each structure, the homodimeric protein is viewed with its 2-fold axis of symmetry vertical and is shown as a ribbon diagram with one subunit gold and the other cyan. The side chains of the active site Asp residues, Asp 25 and Asp 25, as well as the saquinavir in Part b, are shown in ball-and-stick form with C green, N blue, and O red. Note how the hairpin “flaps” at the top of the uncomplexed enzyme have folded down over the inhibitor in the saquinavir complex. Compare these structures with that of the similarly viewed pepsin in Fig. 15-36a. [Part a based on an X-ray structure by Tom Blundell, Birkbeck College, London, U.K., and Part b based on an X-ray structure by Robert Crowther, Hoffmann-LaRoche Ltd., Nutley, New Jersey. PDBids (a) 3PHV and (b) 1HXB.] FIGURE 15-38 O See the Interactive Exercises 7884d_c15.qxd 1/23/03 12:28 PM Page 539 mac18 mac18:df_169:7884D: Section 15–4. Drug Design metrically arranged subunits. Quite possibly HIV-1 protease resembles the putative primordial aspartic protease that, through gene duplication, evolved to form the eukaryotic enzymes (although HIV-1 protease is well suited to the limited amount of genetic information that a virus can carry). Once the structure of HIV-1 protease became available, intensive efforts were mounted in numerous laboratories to find therapeutically effective inhibitors of this enzyme. In this process, 200 X-ray structures and several NMR structures have been reported of HIV-1 protease, its mutants, and the proteases of other retroviruses, both alone and in their complexes with a great variety of inhibitors. Hence, HIV-1 protease is perhaps the most exhaustively structurally studied protein. Comparison of the X-ray structure of HIV-1 protease alone (Fig. 15-38a) with that of its complexes with polypeptidelike inhibitors (e.g., Fig. 15-38b) reveals that, on binding an inhibitor, the hairpin “flaps” covering the “top” of the substrate-binding cleft move down by as much as 7 Å to enclose the inhibitor. Such an inhibitor binds to the 539 2-fold symmetric enzyme in a two-fold pseudosymmetric extended conformation such that the inhibitor interacts with the enzyme much like a strand in a sheet (Fig. 15-39). On the “floor” of the binding cleft, each signature sequence (Asp 25–Thr 26–Gly 27) is located in a loop that is stabilized by a network of hydrogen bonds similar to that observed in eukaryotic aspartic proteases. The inhibitor interacts with the enzyme via a hydrogen bond to the active site residue Asp 25. However, contrary to the case for eukaryotic aspartic proteases (Fig. 15-36b), no X-ray structure of an HIV-1 protease contains a water molecule within hydrogen bonding distance of Asp 25 or Asp 25. On the flap side of the binding cleft, the inhibitor interacts with Gly 48 and Gly 48 and with a water molecule that is not the attacking nucleophile but which mediates the contacts between the flaps and the inhibitor backbone. Although HIV-1 protease specifically cleaves the gag and gag–pol polyproteins at a total of 8 sites (Fig. 15-34b), these sites appear to have little in common except that their immediately flanking residues are nonpolar and mostly bulky. Indeed, binding studies indicate that HIV-1 pro- Flap side S2 S4 P2 P4 Ile 50 N H Gly 48 N H S3 P1 P3 Ile 50 N H Gly 48 C C O H O H H N C N H O H N C C N H N H H N Asp 29 O Asp 29 N H O O O C Gly 27 O HO H N OC O Asp 29 Gly 27 Asp 25 O Asp 25 P3 P1 P2 P4 S3 S1 S2 S4 Arrangement of hydrogen bonds between HIV-1 protease and a modeled substrate. In the nomenclature used here, polypeptide residues in one subunit are assigned primed numbers to differentiate them from the residues of the other subunit; substrate residues on the N-terminal side of the scissile peptide bond are designated P1, P2, P3, … , counting toward the N-terminus; substrate residues on its C-terminal side are FIGURE 15-39 H N C C O O O O H N C C O O Gly 48 N H O O O C S1 designated P1, P2, P3, … , counting toward the C-terminus; and the symbols S1, S2, S3, … , and S1, S2, S3, … , designate the enzyme’s corresponding residue-binding subsites. The scissile peptide bond is marked by arrows. [After Wlodawer, A. and Vondrasek, J., Annu. Rev. Biophys. Biomol. Struct. 27, 257 (1998).] 7884d_c15.qxd 540 2/10/03 9:25 AM Page 540 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis O H N R C N CH CH N N C N H H Indinavir (CrixivanTM) R CH2 CH C N H O R O Reduced Amide H N HO OH R CH CH CH O O N Peptide Bond CH N •H2SO4 O H N S O N N Nelfinavir (ViraceptTM) Hydroxyethylene CH CH N S C CH O N O CH Ph OH H N R O O OH H CH3 N N O H Ph R •CH3SO3H H H O OH NHtBu OH H C CH2 R H N OH Ph H OH N S Ritonavir (NorvirTM) Dihydroxyethylene OH H N O H N CH CH R CH2 C H CH R N N NH2 Comparison of a normal peptide bond (top) to a selection of groups (red) that are isosteres (stereochemical analogs) of the tetrahedral intermediate in reactions catalyzed by aspartic proteases. FIGURE 15-40 H OH O H •CH3SO3H H Saquinavir (InviraseTM) H tease’s specificity arises from the cumulative effects of the interactions between the enzyme and the amino acids in positions P4 through P¿4 . However, three of the peptides cleaved by HIV-1 have either the sequence Phe-Pro or TyrPro, which are sequences that human aspartic proteases do not cleave. Hence, HIV-1 protease inhibitors containing groups that resemble either of these dipeptides would be unlikely to inhibit essential human aspartic proteases. An effective HIV-1 protease inhibitor should resemble a substrate with its scissile peptide replaced by a group that the enzyme cannot cleave. Such a group should, preferably, enhance the enzyme’s affinity for the inhibitor. Mimics of the tetrahedral intermediate (Fig. 15-37), that is, transition state analogs, are likely to do so. Consequently, a variety of such groups (Fig. 15-40) have been investigated in efforts to synthesize therapeutically effective inhibitors of HIV-1 protease. Although HIV-1 protease has high in vitro affinity for its polypeptide-based inhibitors, these substances have poor oral bioavailability (they are degraded by digestive N N O Hydroxyethylamine NHtBu O O O O N O NH2 OH N O S O Ph Amprenavir (AgeneraseTM) Some HIV-1 protease inhibitors that are in clinical use. Note that in addition to its generic (chemical) name, each drug has a proprietary trade name, here in parentheses, under which it is marketed. FIGURE 15-41 proteases) and pharmacokinetics (they do not readily pass through cell membranes.). Consequently, therapeutically effective HIV-1 protease inhibitors must be peptidomimetics (peptide mimics), substances that sterically and perhaps physically, but not chemically, resemble polypeptides. The use of peptidomimetics also permits conformational constraints to be imposed on a drug candidate that would not be present in the corresponding polypeptide. 7884d_c15.qxd 3/24/03 1:01 PM Page 541 mac85 Mac 85:1st shift: 1268_tm:7884d: Chapter Summary As of early 2003, the FDA had approved six HIV-1 protease inhibitors (Fig. 15-41), the first of which, saquinavir, was sanctioned in late 1995. These peptidomimetics have IC50s against HIV in culture ranging from 2 to 60 nM but have little or no activity against human aspartic proteases (KI’s 10 M). They are the first drugs to clearly prolong the lives of AIDS victims. Their development, in each case, was a complex iterative process that required the design, synthesis, and evaluation of numerous related compounds. In several cases, these investigations capitalized on the wealth of experience gained in developing peptidomimetic inhibitors of the aspartic protease renin and in the resulting stockpiles of these compounds. All the FDA-approved HIV-1 protease inhibitors initially cause a rapid and profound decline in a patient’s plasma HIV load, which is often paralleled by immune system recovery. However, as we saw with reverse transcriptase inhibitors, mutant forms of the protease that are resistant to the inhibitor being used arise, usually within 4 to 12 weeks. Moreover, such a mutant protease is likely to be resistant to other HIV-1 protease inhibitors, because all of the HIV-1 protease inhibitors are targeted to the same binding site. This has led to the use of combination therapies in which an HIV-1 protease inhibitor is administered together with one, or more often, two reverse transcriptase inhibitors. This is because any virus that gains resistance to one drug in a regimen will be suppressed by the other drug(s) in that regimen. In addition, the HIV-1 protease inhibitor ritonavir has been shown to be a potent inhibitor of the cytochrome P450 isoforms (CYP3A4,5,7) that metabolize other protease inhibitors and hence is usually prescribed in low dosage as an adjunct to another pro- 541 tease inhibitor to improve the latter’s pharmacokinetics. The plasma virus levels in many patients who were placed on combination therapy rapidly became undetectable and have remained so for several years. This, however, does not constitute a cure: If drug therapy is interrupted, the virus will reappear in the plasma because certain tissues in the body harbor latent viruses that are unaffected by and/or inaccessible to drug therapy. Thus, the presently available anti-HIV medications must be taken for a lifetime. Current anti-HIV therapies are by no means ideal. To maximize their oral bioavailability, some of the different drugs must be taken well before or after a meal but others must be taken with a meal. To minimize the probability of resistant forms of HIV arising, the bioavailability of each drug must be maintained at a certain minimum level and hence each drug must be taken on a rigid schedule. Moreover, these drugs have significant side effects, mainly fatigue, nausea, diarrhea, tingling and numbness with ritonavir, and kidney stones with indinavir. Consequently, numerous AIDS patients fail to take their medications properly, which greatly increases the likelihood that they will develop resistance to these drugs and infect others with drug-resistant viruses. Finally, HIV-1 protease inhibitors, being complex molecules, are difficult to synthesize and therefore are relatively expensive, so that in the developing countries in which AIDS is most prevalent, governments and most individuals cannot afford to purchase these drugs, even if they were to be supplied at cost. It is therefore important that anti-HIV therapies be developed that are easy for patients to comply with, are inexpensive, and ideally, will totally eliminate an HIV infection. CHAPTER SUMMARY 1 Catalytic Mechanisms Most enzymatic mechanisms of catalysis have ample precedent in organic catalytic reactions. Acid- and base-catalyzed reactions occur, respectively, through the donation or abstraction of a proton to or from a reactant so as to stabilize the reaction’s transition state complex. Enzymes often employ ionizable amino acid side chains as general acid–base catalysts. Covalent catalysis involves nucleophilic attack of the catalyst on the substrate to transiently form a covalent bond followed by the electrophilic stabilization of a developing negative charge in the reaction’s transition state. Various protein side chains as well as certain coenzymes can act as covalent catalysts. Metal ions, which are common enzymatic components, catalyze reactions by stabilizing developing negative charges in a manner resembling general acid catalysis. Metal ion–bound water molecules are potent sources of OH ions at neutral pH’s. Metal ions also facilitate enzymatic reactions through the charge shielding of bound substrates. The arrangement of charged groups about an enzymatic active site of low dielectric constant in a manner that stabilizes the transition state complex results in the electrostatic catalysis of the enzymatic reaction. Enzymes catalyze reactions by bringing their substrates into close proximity in reactive orientations. The enzymatic binding of the substrates in a bimolecular reaction arrests their relative motions resulting in a rate enhancement. The preferential enzymatic binding of the transition state of a catalyzed reaction over the substrate is an important rate enhancement mechanism. Transition state analogs are potent competitive inhibitors because they bind to the enzyme more tightly than does the corresponding substrate. 2 Lysozyme Lysozyme catalyzes the hydrolysis of (1S4)-linked poly(NAG–NAM), the bacterial cell wall polysaccharide, as well as that of poly(NAG). According to the Phillips mechanism, lysozyme binds a hexasaccharide so as to distort its D-ring toward the half-chair conformation of the planar oxonium ion transition state. This is followed by cleavage of the C1 ¬ O1 bond between the D- and E-rings as promoted by proton donation from Glu 35. Finally, the resulting oxonium ion transition state is electrostatically stabilized by the nearby carboxyl group of Asp 52 so that the E-ring can be replaced by OH to form the hydrolyzed product. The roles of Glu 35 and Asp 52 in lysozyme catalysis have been verified through mutagenesis studies. Similarly, structural and binding studies indicate that strain is of major catalytic importance in the lysozyme mechanism. However, mass spectrometry and X-ray studies have demonstrated that the 7884d_c15.qxd 3/24/03 1:01 PM Page 542 mac85 Mac 85:1st shift: 1268_tm:7884d: 542 Chapter 15. Enzymatic Catalysis lysozyme reaction proceeds via a covalent glycosyl–enzyme intermediate involving Asp52 rather than by the noncovalently bound oxonium ion intermediate postulated by the Phillips mechanism. 3 Serine Proteases Serine proteases constitute a widespread class of proteolytic enzymes that are characterized by the possession of a reactive Ser residue. The pancreatically synthesized digestive enzymes trypsin, chymotrypsin, and elastase are sequentially and structurally related but have different side chain specificities for their substrates. All have the same catalytic triad, Asp 102, His 57, and Ser 195, at their active sites. The differing side chain specificities of trypsin and chymotrypsin depend in a complex way on the structures of the loops that connect the walls of the specificity pocket, as well as on the charge of the side chain at the base of the specificity pocket. Subtilisin, serine carboxypeptidase II, and ClpP are unrelated serine proteases that have essentially the same active site geometry as do the pancreatic enzymes. Catalysis in serine proteases is initiated by the nucleophilic attack of the active Ser on the carbonyl carbon atom of the scissile peptide to form the tetrahedral intermediate, a process that may be facilitated by the formation of a low-barrier hydrogen bond between Asp 102 and His 57. The tetrahedral intermediate, which is stabilized by its preferential binding to the enzyme’s active site, then decomposes to the acyl–enzyme intermediate under the impetus of proton donation from the Asp 102polarized His 57. After the replacement of the leaving group by solvent H2O, the catalytic process is reversed to yield the second product and the regenerated enzyme. The Asp 102–His 57 couple therefore functions in the reaction as a proton shuttle. The active Ser is not unusually reactive but is ideally situated to nucleophilically attack the activated scissile peptide. The X-ray structure of the trypsin–BPTI complex indicates the existence of the tetrahedral intermediate, whereas X-ray structures of a complex of elastase with the heptapeptide BCM7 have visualized both the acyl–enzyme intermediate and the tetrahedral intermediate. The pancreatic serine proteases are synthesized as zymogens to prevent pancreatic self-digestion. Trypsinogen is activated by a single proteolytic cleavage by enteropeptidase. The resulting trypsin similarly activates trypsinogen as well as chymotrypsinogen, proelastase, and other pancreatic digestive enzymes. Trypsinogen’s catalytic triad is structurally intact. The zymogen’s low catalytic activity arises from a distortion of its specificity pocket and oxyanion hole, so that it is unable to productively bind substrate or preferentially bind the catalytic reaction’s transition state. 4 Drug Design Drugs act by binding to and thereby modifying the functions of receptors. Many promising drug candidates, which are known as lead compounds, have been found by methods in which a large number of compounds are tested for drug efficacy in an assay that is a suitable surrogate of the disease/condition under consideration. Lead compounds are then chemically manipulated in the search for compounds with improved drug efficacy. Structure–activity relationships (SARs) and quantitative structure–activity relationships (QSARs) are useful tools in this endeavor. Structure-based drug design uses the X-ray and NMR structures of drug candidates in complex with their target proteins, together with a variety of molecular modeling tools, to guide the search for improved drug candidates. However, the advent of combinatorial chemistry and high-throughput screening procedures has extended the “make-many-compounds-andsee-what-they-do” approaches to drug discovery. In order to reach their target receptors, drugs must have favorable pharmacokinetics, that is, they must readily traverse numerous physical barriers in the body, avoid chemical transformation by enzymes, and not be excreted too rapidly. Most useful drugs are neither too lipophilic nor too hydrophilic so that they can both gain access to the necessary membranes and pass through them. Drug toxicity, dosage, efficacy, and the nature of rare adverse reactions are determined through extensive and carefully designed clinical trials. Most drugs are metabolically cleared through oxidative hydroxylation by one of the 100 cytochrome P450 isozymes. This permits the hydroxylated drugs to be enzymatically conjugated to polar groups such as glucuronic acid and glycine, which increases their rates of excretion by the kidneys. Drug–drug interactions are frequently mediated by cytochromes P450. Polymorphisms among cytochromes P450 are often responsible for the variations among individuals in their response to a given drug, including adverse reactions. The formulation of HIV-1 protease inhibitors to control HIV infections is one of the major triumphs of modern drug discovery methods. HIV are retroviruses that attack specific immune system cells and thereby degrade the immune system over a period of several years to the point that it is no longer able defend against opportunistic infections. HIV-1 protease functions to cleave the polyproteins in immature HIV-1 virions that have budded out from a host cell, thus generating the mature, infectious form. HIV-1 protease is an aspartic protease that, as eukaryotic aspartic proteases such as pepsin, uses its two active site Asp residues to activate its bound lytic water molecule as the nucelophile that attacks and thereby cleaves specific peptide bonds in the substrate polyprotein. All of the FDA-approved peptidomimetic inhibitors of HIV1 protease cause a rapid and profound decrease in plasma HIV levels, although they do not entirely eliminate the virus. They are used in combination with reverse transcriptase inhibitors to minimize the ability of the rapidly mutating HIV to evolve drug-resistant forms. REFERENCES GENERAL Bender, M.L., Bergeron, R.J., and Komiyama, M., The Bioorganic Chemistry of Enzymatic Catalysis, Wiley (1984). Fersht, A., Structure and Mechanism in Protein Science, Freeman (1999). Jencks, W.P., Catalysis in Chemistry and Enzymology, Dover (1987). [A classic and, in many ways, still current work.] Walsh, C., Enzymatic Reaction Mechanisms, Freeman (1979). [A compendium of enzymatic reactions.] 7884d_c15.qxd 1/23/03 12:28 PM Page 543 mac18 mac18:df_169:7884D: References CATALYTIC MECHANISMS Atkins, W.M. and Sligar, S.G., Protein engineering for studying enzyme catalytic mechanism, Curr. Opin. Struct. Biol. 1, 611–616 (1991). Bruice, T.C., Some pertinent aspects of mechanism as determined with small molecules, Annu. Rev. Biochem. 45, 331–373 (1976). Bruice, T.C. and Benkovic, S.J., Chemical basis for enzyme catalysis, Biochemistry 39, 6267–6274 (2000); and Bruice, T.C. and Lightstone, F.C., Ground state and transition state contributions to the rates of intramolecular and enzymatic reactions, Acc. Chem. Res. 32, 127–136 (1999). Christianson, D.W. and Cox, J.D., Catalysis by metal-activated hydroxide in zinc and manganese metalloenzymes, Annu. Rev. 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Stroud, R.M., Kossiakoff, A.A., and Chambers, J.L., Mechanism of zymogen activation, Annu. Rev. Biophys. Bioeng. 6, 177–193 (1977). Wang, J., Hartling, J.A., and Flanagan, J.M., The structure of ClpP at 2.3 Å resolution suggests a model for ATP-dependent proteolysis, Cell 91, 447–456 (1997). Wilmouth, R.C., Edman, K., Neutze, R., Wright, P.A., Clifton, I.J., Schneider, T.R., Schofield, C.J., and Hadju, J., X-Ray snapshots of serine protease catalysis reveals a tetrahedral intermediate, Nature Struct. Biol. 8, 689–694 (2001); and Wilmouth, R.C., Clifton, I.J., Robinson, C.V., Roach, P.L., Aplin, R.T., Westwood, N.J., Hadju, J., and Schofield, C.J., Structure of a specific acyl-enzyme complex formed between -casomorphin-7 and porcine pancreatic elastase, Nature Struct. Biol. 4, 456–461 (1997). DRUG DISCOVERY Debouck, C. and Metcalf, B., The impact of genomics on drug discovery, Annu. Rev. Pharmacol. Toxicol. 40, 193–208 (2000). Gordon, E.M. and Kerwin, J.F., Jr. (Eds.), Combinatorial Chemistry and Molecular Diversity in Drug Discovery, Wiley-Liss (1998). Gringauz, A., Introduction to Medicinal Chemistry, Wiley-VCH (1997). Harman, J.G., Limbird, L.E., Molinoff, P.B., Ruddon, R.W., and Gilman, A.G. (Eds.), Goodman & Gilman’s The Pharmacologic Basis of Therapeutics (9th ed.), McGraw-Hill (2000). Ingelman-Sundberg, M., Oscarson, M., and McLellan, R.A., Polymorphic human cytochrome P450 enzymes: An opportunity for individualized drug treatment, Trends Pharmacol. Sci. 20, 342–349 (1999). Katzung, B.G. (Ed.), Basic & Clinical Pharmacology (7th ed.), Appleton & Lange (1998). Marrone, T.J., Briggs, J.M., and McCammon, J.A., Structurebased drug design: Computational advances, Annu. Rev. Pharmacol. Toxicol. 37, 71–90 (1997). Mycek, M.J., Harvey, R.A., and Champe, P.C., Lippincotts Illustrated Reviews: Pharmacology (2nd ed.), Lippincott– Raven Publishers (1997). Navia, M.A. and Murcko, M.A., Use of structural information in drug design, Curr. Opin. Struct. Biol. 2, 202–210 (1992). Ohlstein, E.H., Ruffolo, R.R., Jr., and Elliott, J.D., Drug discovery in the next millennium, Annu. Rev. Pharmacol. Toxicol. 40, 177–191 (2000). Patrick, G.L, An Introduction to Medicinal Chemistry, Oxford University Press (1995). Smith, D.A. and van der Waterbeemd, H., Pharmacokinetics and metabolism in early drug design, Curr. Opin. Chem. Biol. 3, 373–378 (1999). Terrett, N.O., Combinatorial Chemistry, Oxford University Press (1998). Walsh, G., Biopharmaceuticals: Biochemistry and Biotechnology, Wiley (1998). White, R.E., High-throughput screening in drug metabolism and pharmacokinetic support of drug discovery, Annu. Rev. Pharmacol. Toxicol. 40, 133–157 (2000). Wong, L.-L., Cytochrome P450 monooxygenases, Curr. Opin. Chem. Biol. 2, 263–268 (1998). HIV-1 PROTEASE AND OTHER ASPARTIC PROTEASES Davies, D.R., The structure and function of the aspartic proteases, Annu. Rev. Biophys. Biophys. Chem. 19, 189–215 (1990). Erickson, J.W. and Burt, S.K., Structural mechanisms of HIV drug resistance, Annu. Rev. Pharmacol. Toxicol. 36, 545–571 (1996). Flexner, C., Dual protease inhibitor therapy in HIV-infected patients: Pharmacological rationale and clinical benefits, Annu. Rev. Pharmacol. Toxicol. 40, 649–674 (2000). Kling, J., Blocking HIV’s “scissors,” Modern Drug Discovery 3(2), 37–45 (2000). Meeks, T.D., Catalytic mechanisms of the aspartic proteases, in Sinnott, M. (Ed.), Comprehensive Biological Catalysis, Vol. 1, pp. 327–344, Academic Press (1998). Richman, D.D., HIV chemotherapy, Nature 410, 995–1001 (2001). Tomesselli, A.G., Thaisrivongs, S., and Heinrikson, R. L., Discovery and design of HIV protease inhibitors as drugs for treatment of AIDS, Adv. Antiviral Drug Design 2, 173–228 (1996). Turner, B.G. and Summers, M.F., Structural biology of HIV, J. Mol. Biol. 285, 1–32 (1999). [A review.] Wilk, T. and Fuller, S.D., Towards the structure of human immunodeficiency virus: Divide and conquer? Curr. Opin. Struct. Biol. 9, 231–243 (1999). Wlodawer, A. Rational approach to AIDs drug design through structural biology, Annu. Rev. Med. 53, 595–614 (2001); and Wlodawer, A. and Vondrasek, J., Inhibitors of HIV-1 protease: A major success of structure-assisted drug design, Annu. Rev. Biophys. Biomol. Struct. 27, 249–284 (1998). PROBLEMS 1. Explain why -pyridone is not nearly as effective a catalyst for glucose mutarotation as is -pyridone. What about -pyridone? 2. RNA is rapidly hydrolyzed in alkaline solution to yield a mixture of nucleotides whose phosphate groups are bonded to either the 2 or the 3 positions of the ribose residues. DNA, which 7884d_c15.qxd 1/23/03 12:29 PM Page 545 mac18 mac18:df_169:7884D: Problems lacks RNA’s 2 OH groups, is resistant to alkaline degradation. Explain. 3. Carboxypeptidase A, a Zn2-containing enzyme, hydrolyzes the C-terminal peptide bonds of polypeptides (Section 7-1A). In the enzyme–substrate complex, the Zn2 ion is coordinated to three enzyme side chains, the carbonyl oxygen of the scissile peptide bond, and a water molecule. A plausible model for the enzyme’s reaction mechanism that is consistent with Xray and enzymological data is diagrammed in Fig. 15-42. What are the roles of the Zn2 ion and Glu 270 in this mechanism? 545 4. In the following lactonization reaction, C C R R O O OH CH2 CH2 C COO R R R R the relative reaction rate when R CH 3 is 3.4 1011 times that when R H. Explain. *5. Derive the analog of Eq. [15.11] for an enzyme that catalyzes the reaction: ABSP Assume the enzyme must bind A before it can bind B: E A B ∆ EA B ∆ EAB S EP CO2– CHR O Glu 270 C NH R O– C H O O 2+ H Zn attack of water CHR C NH R O– C H O– O Zn2+ H 8. Wolfenden has stated that it is meaningless to distinguish between the “binding sites” and the “catalytic sites” of enzymes. Explain. 10. In light of the information given in this chapter, why are enzymes such large molecules? Why are active sites almost always located in clefts or depressions in enzymes rather than on protrusions? CO2– O 7. Suggest a transition state analog for proline racemase that differs from those discussed in the text. Justify your suggestion. 9. Explain why oxalate (OOCCOO) is an inhibitor of oxaloacetate decarboxylase. Michaelis complex Glu 270 6. Explain, in thermodynamic terms, why an “enzyme” that stabilizes its Michaelis complex as much as its transition state does not catalyze a reaction. Tetrahedral intermediate 11. Predict the effects on lysozyme catalysis of changing Phe 34, Ser 36, and Trp 108 to Arg, assuming that this change does not significantly alter the structure of the protein. *12. The incubation of (NAG)4 with lysozyme results in the slow formation of (NAG)6 and (NAG)2. Propose a mechanism for this reaction. What aspect of the Phillips mechanism is established by this reaction? 13. How would the lysozyme binding affinity of the following (1S4)-linked tetrasaccharide scissle bond scission CO2– CHR O C H R C O O H Zn2+ Enzyme-product complex FIGURE 15-42 NAG O H NHCOCH3 compare with that of NAG–NAM–NAG–NAM? Explain. H + O– NAM H N Glu 270 H NAG CH2OH O H H Mechanism of carboxypeptidase A. 14. A major difficulty in investigating the properties of the pancreatic serine proteases is that these enzymes, being proteins themselves, are self-digesting. This problem is less severe, however, for solutions of chymotrypsin than it is for solutions of trypsin or elastase. Explain. 15. The comparison of the active site geometries of chymotrypsin and subtilisin under the assumption that their similarities have catalytic significance has led to greater mechanistic understanding of both these enzymes. Discuss the validity of this strategy. 7884d_c15.qxd 546 1/23/03 12:29 PM Page 546 mac18 mac18:df_169:7884D: Chapter 15. Enzymatic Catalysis 16. Benzamidine (KI 1.8 105M) and leupeptin (KI 1.8 107M) O CH3C O Leu Leu NH CH CH (CH2)3 NH C H2N C NH2+ H2N Benzamidine NH2+ Leupeptin are both specific competitive inhibitors of trypsin. Explain their mechanisms of inhibition. Design leupeptin analogs that inhibit chymotrypsin and elastase. 17. Trigonal boronic acid derivatives have a high tendency to form tetrahedral adducts. 2-Phenylethyl boronic acid OH CH2 CH2 B OH 2–Phenylethyl boronic acid is an inhibitor of subtilisin and chymotrypsin. Indicate the structure of these enzyme–inhibitor complexes. 18. Tofu (bean curd), a high-protein soybean product that is widely consumed in China and Japan, is prepared in such a way as to remove the trypsin inhibitor present in soybeans. Explain the reason(s) for this treatment. 19. Explain why mutating all three residues of trypsin’s catalytic triad has essentially no greater effect on the enzyme’s catalytic rate enhancement than mutating only Ser 195. 20. Explain why chymotrypsin is not self-activating as is trypsin. 21. Does Lipinski’s “rule of five” predict that a hexapeptide would be a therapeutically effective drug? Explain. 22. The preferred antidote for acetaminophen overdose is N-acetylcysteine. Explain why the administration of this substance, which must occur within 8 to 16 hours of the overdose, is an effective treatment. 23. Why would the activation of HIV-1 protease before the virus buds from its host cell be disadvantageous to the virus? Explain.