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Enzymes IV Andy Howard Introductory Biochemistry 5 November 2008 Biochem: Enzymes IV 11/5/2009 More about mechanisms Many enzymatic mechanisms involve either covalent catalysis or acid-base interactions We’ll give some examples of several mechanistic approaches 11/5/2009 Biochem: Enzymes IV P. 2 of 62 Mechanism Topics Enzyme dynamics Enzyme chemistry Diffusion-controlled Reactions Binding Modes of Catalysis Induced-fit Redox reactions Transition-state binding: review 11/5/2009 Biochem: Enzymes IV Induced fit Tight binding of ioinic intermediates Specific mechanisms Serine proteases Cysteinyl proteases Lysozyme TIM P. 3 of 62 The protein moves as well! Changes to active-site conformation: Help with substrate binding Position the catalytic groups Induce formation of an NAC Help to break or make bonds Facilitate conversion of S to P Sometimes involve networks of concerted amino acid changes 11/5/2009 Biochem: Enzymes IV P. 4 of 62 Ionic reactions Define them as reactions that involve charged, or at least polar, intermediates Typically 2 reactants Electron rich (nucleophilic) reactant Electron poor (electrophilic) reactant Conventional to describe reaction as attack of nucleophile on electrophile Drawn with nucleophile donating electron(s) to electrophile 11/5/2009 Biochem: Enzymes IV P. 5 of 62 Reactive sidechains in a.a.’s AA Group Charge @pH=7 Asp —COO-1 Glu —COO-1 His Imidazole ~0 Cys —CH2SH ~0 Tyr Phenol 0 Lys NH3+ +1 Arg guanadinium +1 Ser —CH2OH 0 Functions Cation binding, H+ transfer Same as above Proton transfer Covalent binding of acyl gps H-bonding to ligands Anion binding, H+ transfer Anion binding See cys 11/5/2009 Biochem: Enzymes IV P. 6 of 62 Generalizations about activesite amino acids Typical enzyme has 2-6 key catalytic residues His, asp, arg, glu, lys account for 64% Remember: pKa values in proteins sometimes different from those of isolated aa’s Frequency overall Frequency in catalysis 11/5/2009 Biochem: Enzymes IV P. 7 of 62 Attack on Acyl Group Transfer of an acyl group: section 14.6 Nucleophile Y attacks carbonyl carbon, forming tetrahedral intermediate X- is leaving group 11/5/2009 Biochem: Enzymes IV P. 8 of 62 Direct Displacement Attacking group adds to face of atom opposite to leaving group Transition state can have five ligands; This is inherently less stable than other attacks, but it can still work 11/5/2009 Biochem: Enzymes IV P. 9 of 62 Cleavage Reactions Both electrons stay with one atom Covalent bond produces carbanion: R3—C—H R3—C:- + H+ Covalent bond produces carbocation: R3—C—H R3—C+ + :H- One electron stays with each product Both end up as radicals R1O—OR2 R1O• + •OR2 Radicals are highly reactive— some more than others 11/5/2009 Biochem: Enzymes IV P. 10 of 62 Cleavages by base Simple cleavage: —X—H + :B —X:- + H—B+ This works if X=N,O; sometimes C Removal of proton from H2O to cleave C-X: O O O —C—N —C—OH + HN —C—N HO O H H :B H—B+ 11/5/2009 Biochem: Enzymes IV :B P. 11 of 62 Cleavage by acid Covalent bond may break more easily if one of its atoms is protonated Formation of unstable intermediate, R-OH2+, accelerates the reaction Example: R+ + OH- R—OH R—OH2+ (Slow) (Fast) R+ + H2O 11/5/2009 Biochem: Enzymes IV P. 12 of 62 Low-barrier H-bonds Ordinary H-bonds buy us 10-30 kJ mol-1 O-O separation = 0.28nm (similar for O-N) O-H = 0.1 nm so H…O distance is 0.18nm As the O’s get closer to each other, the bond order gets closer to 0.5 for both We than have an O-O distance ~ 0.22 nm & much stronger (60 kJ mol-1) interaction pKa for the two heteroatoms must be nearly equal for this to happen Several mechanisms employ these 11/5/2009 Biochem: Enzymes IV P. 13 of 62 Rates often depend on pH If an amino acid that is necessary to the mechanism changes protonation state at a particular pH, then the reaction may be allowed or disallowed depending on pH Two ionizable residues means there may be a narrow pH optimum for catalysis 11/5/2009 Biochem: Enzymes IV P. 14 of 62 Papain as an example 1 relative reaction rate Papain pH-rate profile Cys-25 His-159 0 2 3 4 5 6 7 8 9 10 11 pH 11/5/2009 Biochem: Enzymes IV P. 15 of 62 Covalent catalysis Reactive side-chain can be a nucleophile or an electrophile, but nucleophile is more common A—X + E X—E + A X—E + B B—X + E Example: sucrose phosphorylase Net reaction: Sucrose + Pi Glucose 1-P + fructose Fructose=A, Glucose=X, Phosphate=B 11/5/2009 Biochem: Enzymes IV P. 16 of 62 Oxidation-Reduction Reactions Commonplace in biochemistry: EC 1 Oxidation is a loss of electrons Reduction is the gain of electrons In practice, often: oxidation is decrease in # of C-H bonds; reduction is increase in # of C-H bonds Intermediate electron acceptors and donors are organic moieties or metals Ultimate electron acceptor in aerobic organisms is usually dioxygen (O2) 11/5/2009 Biochem: Enzymes IV P. 17 of 62 Biological redox reactions Generally 2-electron transformations Often involve alcohols, aldehydes, ketones, carboxylic acids, C=C bonds: R1R2CH-OH + X R1R2C=O + XH2 R1HC=O + X + OH- R1COO- + XH2 X is usually NAD, NADP, FAD, FMN A few biological redox systems involve metal ions or Fe-S complexes Usually reduced compounds are higher-energy than the corresponding oxidized compounds 11/5/2009 Biochem: Enzymes IV P. 18 of 62 One-electron redox reactions FMN, FAD, some metal ions can be oxidized or reduced one electron at a time With organic cofactors this generally leaves a free radical in each of two places Subsequent reactions get us back to an even number of electrons 11/5/2009 Biochem: Enzymes IV P. 19 of 62 Diffusion-controlled reactions Some enzymes are so efficient that the limiting factor in completion of the reaction is diffusion of the substrates into the active site: These are diffusion-controlled reactions. Ultra-high turnover rates: kcat ~ 109 s-1. We can describe kcat / Km as catalytic efficiency of an enzyme. A diffusioncontrolled reaction will have a catalytic efficiency on the order of 108 M-1s-1. 11/5/2009 Biochem: Enzymes IV P. 20 of 62 Binding modes: proximity We describe enzymatic mechanisms in terms of the binding modes of the substrates (or, more properly, the transition-state species) to the enzyme. One of these involves the proximity effect, in which two (or more) substrates are directed down potential-energy gradients to positions where they are close to one another. Thus the enzyme is able to defeat the entropic difficulty of bringing substrates together. 11/5/2009 Biochem: Enzymes IV William Jencks P. 21 of 62 Binding modes: efficient transition-state binding Transition state fits even better (geometrically and electrostatically) in the active site than the substrate would. This improved fit lowers the energy of the transition-state system relative to the substrate. Best competitive inhibitors of an enzyme are those that resemble the transition state rather than the substrate or product. 11/5/2009 Biochem: Enzymes IV P. 22 of 62 Proline racemase Pyrrole-2-carboyxlate resembles planar transition state 11/5/2009 Biochem: Enzymes IV P. 23 of 62 Yeast aldolase Phosphoglycolohydroxamate binds much like the transition state to the catalytic Zn2+ 11/5/2009 Biochem: Enzymes IV P. 24 of 62 Adenosine deaminase with transition-state analog Transition-state analog: Ki~10-8 * substrate Km Wilson et al (1991) Science 252: 1278 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 11/5/2009 Biochem: Enzymes IV P. 25 of 62 ADA transition-state analog 1,6 hydrate of purine ribonucleoside binds with KI ~ 3*10-13 M 11/5/2009 Biochem: Enzymes IV P. 26 of 62 Induced fit Refinement on original Emil Fischer lock-and-key notion: both the substrate (or transitionstate) and the enzyme have flexibility Binding induces conformational changes 11/5/2009 Biochem: Enzymes IV P. 27 of 62 Example: hexokinase Glucose + ATP Glucose-6-P + ADP Risk: unproductive reaction with water Enzyme exists in open & closed forms Glucose induces conversion to closed form; water can’t do that Energy expended moving to closed form 11/5/2009 Biochem: Enzymes IV P. 28 of 62 Hexokinase structure Diagram courtesy E. Marcotte, UT Austin 11/5/2009 Biochem: Enzymes IV P. 29 of 62 Tight binding of ionic intermediates Quasi-stable ionic species strongly bound by ion-pair and H-bond interactions Similar to notion that transition states are the most tightly bound species, but these are more stable 11/5/2009 Biochem: Enzymes IV P. 30 of 62 Serine protease mechanism Only detailed mechanism that we’ll ask you to memorize One of the first to be elucidated Well studied structurally Illustrates many other mechanisms Instance of convergent and divergent evolution 11/5/2009 Biochem: Enzymes IV P. 31 of 62 The reaction Hydrolytic cleavage of peptide bond Enzyme usually works on esters too Found in eukaryotic digestive enzymes and in bacterial systems Widely-varying substrate specificities Some proteases are highly specific for particular aas at position 1, 2, -1, . . . Others are more promiscuous CH NH R1 NH C C NH CH 11/5/2009 OBiochem: EnzymesRIV -1 P. 32 of 62 Mechanism Active-site serine —OH … Without neighboring amino acids, it’s fairly non-reactive becomes powerful nucleophile because OH proton lies near unprotonated N of His This N can abstract the hydrogen at nearneutral pH Resulting + charge on His is stabilized by its proximity to a nearby carboxylate group on an aspartate side-chain. 11/5/2009 Biochem: Enzymes IV P. 33 of 62 Catalytic triad The catalytic triad of asp, his, and ser is found in an approximately linear arrangement in all the serine proteases, all the way from non-specific, secreted bacterial proteases to highly regulated and highly specific mammalian proteases. 11/5/2009 Biochem: Enzymes IV P. 34 of 62 Diagram of first three steps 11/5/2009 Biochem: Enzymes IV P. 35 of 62 Diagram of last four steps Diagrams courtesy University of Virginia 11/5/2009 Biochem: Enzymes IV P. 36 of 62 Chymotrypsin as example Catalytic Ser is Ser195 Asp is 102, His is 57 Note symmetry of mechanism: steps read similarly L R and R L Diagram courtesy of Anthony Serianni, University of Notre Dame 11/5/2009 Biochem: Enzymes IV P. 37 of 62 Oxyanion hole When his-57 accepts proton from Ser-195: it creates an R—O- ion on Ser sidechain In reality the Ser O immediately becomes covalently bonded to substrate carbonyl carbon, moving - charge to the carbonyl O. Oxyanion is on the substrate's oxygen Oxyanion stabilized by additional interaction in addition to the protonated his 57: main-chain NH group from gly 193 H-bonds to oxygen atom (or ion) from the substrate, further stabilizing the ion. 11/5/2009 Biochem: Enzymes IV P. 38 of 62 Oxyanion hole cartoon Cartoon courtesy Henry Jakubowski, College of St.Benedict / St.John’s University 11/5/2009 Biochem: Enzymes IV P. 39 of 62 Modes of catalysis in serine proteases Proximity effect: gathering of reactants in steps 1 and 4 Acid-base catalysis at histidine in steps 2 and 4 Covalent catalysis on serine hydroxymethyl group in steps 2-5 So both chemical (acid-base & covalent) and binding modes (proximity & transition-state) are used in this mechanism 11/5/2009 Biochem: Enzymes IV P. 40 of 62 Specificity Active site catalytic triad is nearly invariant for eukaryotic serine proteases Remainder of cavity where reaction occurs varies significantly from protease to protease. In chymotrypsin hydrophobic pocket just upstream of the position where scissile bond sits This accommodates large hydrophobic side chain like that of phe, and doesn’t comfortably accommodate hydrophilic or small side chain. Thus specificity is conferred by the shape and electrostatic character of the site. 11/5/2009 Biochem: Enzymes IV P. 41 of 62 Chymotrypsin active site Comfortably accommodates aromatics at S1 site Differs from other mammalian serine proteases in specificity Diagram courtesy School of Crystallography, Birkbeck College 11/5/2009 Biochem: Enzymes IV P. 42 of 62 Divergent evolution Ancestral eukaryotic serine proteases presumably have differentiated into forms with different side-chain specificities Chymotrypsin is substantially conserved within eukaryotes, but is distinctly different from elastase 11/5/2009 Biochem: Enzymes IV P. 43 of 62 iClicker quiz! Why would the nonproductive hexokinase reaction H2O + ATP -> ADP + Pi be considered nonproductive? (a) Because it needlessly soaks up water (b) Because the enzyme undergoes a wasteful conformational change (c) Because the energy in the high-energy phosphate bond is unavailable for other purposes (d) Because ADP is poisonous (e) None of the above 11/5/2009 Biochem: Enzymes IV P. 44 of 62 iClicker quiz, question 2: Why are proteases often synthesized as zymogens? (a) Because the transcriptional machinery cannot function otherwise (b) To prevent the enzyme from cleaving peptide bonds outside of its intended realm (c) To exert control over the proteolytic reaction (d) None of the above 11/5/2009 Biochem: Enzymes IV P. 45 of 62 Question 3: what would bind tightest in the TIM active site? (a) DHAP (substrate) (b) D-glyceraldehyde (product) (c) 2-phosphoglycolate (Transition-state analog) (d) They would all bind equally well 11/5/2009 Biochem: Enzymes IV P. 46 of 62 Convergent evolution Reappearance of ser-his-asp triad in unrelated settings Subtilisin: externals very different from mammalian serine proteases; triad same 11/5/2009 Biochem: Enzymes IV P. 47 of 62 Subtilisin mutagenesis Substitutions for any of the amino acids in the catalytic triad has disastrous effects on the catalytic activity, as measured by kcat. Km affected only slightly, since the structure of the binding pocket is not altered very much by conservative mutations. An interesting (and somewhat non-intuitive) result is that even these "broken" enzymes still catalyze the hydrolysis of some test substrates at much higher rates than buffer alone would provide. I would encourage you to think about why that might be true. 11/5/2009 Biochem: Enzymes IV P. 48 of 62 Cysteinyl proteases Ancestrally related to ser proteases? Cathepsins, caspases, papain Contrasts: Cys —SH is more basic than ser —OH Residue is less hydrophilic S- is a weaker nucleophile than O- 11/5/2009 Biochem: Enzymes IV Diagram courtesy of Mariusz Jaskolski, U. Poznan P. 49 of 62 Papain active site Diagram courtesy Martin Harrison, Manchester University 11/5/2009 Biochem: Enzymes IV P. 50 of 62 Hen egg-white lysozyme Antibacterial protectant of growing chick embryo Hydrolyzes bacterial cell-wall peptidoglycans “hydrogen atom of structural biology” QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. HEWL PDB 2vb1 0.65Å 15 kDa Commercially available in pure form Easy to crystallize and do structure work Available in multiple crystal forms Mechanism is surprisingly complex (14.7) 11/5/2009 Biochem: Enzymes IV P. 51 of 62 Mechanism of lysozyme Strain-induced destabilization of substrate makes the substrate look more like the transition state Long arguments about the nature of the intermediates Accepted answer: covalent intermediate between D52 and glycosyl C1 (14.39B) 11/5/2009 Biochem: Enzymes IV P. 52 of 62 The controversy 11/5/2009 Biochem: Enzymes IV P. 53 of 62 Triosephosphate isomerase (TIM) dihydroxyacetone phosphate glyceraldehyde-3-phosphate Glyc-3-P DHAP 11/5/2009 Biochem: Enzymes IV Km=10µM kcat=4000s-1 kcat/Km=4*108M-1s-1 P. 54 of 62 TIM mechanism DHAP carbonyl H-bonds to neutral imidazole of his-95; proton moves from C1 to carboxylate of glu165 Enediolate intermediate (C—O- on C2) Imidazolate (negative!) form of his95 interacts with C1—O-H) glu165 donates proton back to C2 See Fort’s treatment (http://chemistry.umeche.maine.edu/ CHY431/Enzyme3.html) 11/5/2009 Biochem: Enzymes IV P. 55 of 62