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(1) Kinetic Studies -the type of information available from kinetic studies can be seen from the Table 5.1 (a) Variation of substrate concentration--steady state kinetic studies support the proposal that one substrate reactions proceed via the formation and decay of one or more E-S complexes but cannot give any indication of the sequence of such complexes. Thus, need additional information from product inhibition, substrate binding in order to distinguish between mechanisms in which a ternary complex is formed in an ordered or a random fashion (b) Variation of substrate structure--can learn a great deal about the general features of enzyme active sites by correlating the rates of reactions with the structures of the substrates -compare the rates of hydrolysis of a large number of amide derivatives of amino acids has made it possible to demonstrate that chymotrypsin has a strong preference for substrates containing aromatic or bulky hydrophobic R groups cf. elastase which has a strong preference for substrates containing small hydrophobic R groups -the substrate-binding sites of these enzymes must contain features that account for the observed specificities -exhaustive studies on specificity of papain towards synthetic peptide substrates 90 -several subsites on the enzyme and that subsite S2 interacts specifically with the L-Phe side chain -further work showed that subsite S1' is stereospecific for Lamino acids with a preference for the hydrophobic side chains of Leu and Trp (c) Reversible inhibition--study of inhibition of enzymecatalyzed rxns can give information on the structures of active sites -most likely explanation for competitive inhibition is that the substrate and inhibitor bind to the same site on the enzyme -compare the structure of the substrate and inhibitor and deduce the essential structural features of these molecules that are involved in their binding to the active site -for papain, the tripeptide, Ala-Phe-Arg is a powerful competitive inhibitor of the enzyme since it occupied the subsites S3, S2, and S1 and could not therefore undergo hydrolysis -competitive inhibitors are used in X-ray crystallographic studies where it is usually difficult to study the E-S complex directly (d) Variation of pH--catalytic activity of many enzymes is markedly dependent on pH -many reasons but foremost is the ionization of the amino acid side chains that are involved in the catalytic mechanism 91 -plots of rxn rate versus pH makes it possible to deduce the pKa values of the side chains involved in catalysis -caveat: microenvironment of a side chain can shift the pKa of the side chain by up to four units from the value for the free amino acid -approach used to implicate two His side chains as being involved in the catalytic mechanisms of ribonuclease, later confirmed by X-ray crystallography (e) Pre-steady state kinetics--can be used to detect enzymecontaining complexes in a rxn and to determine the rates of formation and decay of such complexes -the concn of enzyme more closely approaches that of the substrates than is the case with steady state studies but usually requires special techniques to achieve rapid mixing and rapid detection of the changes occurring -eg., chymotrypsin catalyzed hydrolysis of p-nitrophenylacetate -production of p-nitrophenol shows a burst phase (the size of which corresponds to approximately 1 mol per mol enzyme) followed by a slower steady state rate -consistent with a mechanism where a fast step, corresponding to the formation of acyl enzyme and release of p-nitrophenol, is followed by a slow step, corresponding to the rate of hydrolysis of the acyl enzyme to regenerate enzyme 92 (2) Detection of Intermediates--a direct method for obtaining information about the pathway of a rxn is to detect any intermediates that may be involved in the rxn -may be sufficiently stable to be isolated and characterized -may be inferred to exist from spectroscopic studies -rates of formation and decay of any presumed intermediate must be consistent with the overall rate of the rxn -the rate of breakdown of the acyl enzyme intermediate of chymotrypsin is very slow at acid pH, so the intermediate can be isolated (and crystallized) if the enzyme and ester substrate are mixed and the pH is rapidly lowered -X-ray crystallographic work has shown that it is the side chain of Ser 195 that becomes acylated (3) X-ray Crystallographic Studies--the collection of diffraction data takes several hours by which time conversion of substrate to product usually has occurred; however, a number of approaches used to obtain crystallographic data on the active site (i) examine the active complex in the case of a one substrate rxn when the equilibrium lies very much to one side (ii) study the enzyme in the presence of a very poor substrate or competitive inhibitor 93 (iii) examine unstable complexes at low temperature where the rate of decomposition of such complexes will be slowed (iv) use synchrotron radiation (radiation produced during the acceleration of charged particles) to reduce data collection time to seconds -advantages: direct location of active site and information on the nature of the side chain involved in the catalytic mechanism; examine extent of any structural change accompanying the binding of substrate -limitations: can only provide a static picture of structure; danger of differences in structure because of the high ionic strength conditions required for crystallization of protein-however, many enzymes are active in the crystal state (4) Chemical Modification of Amino Acid Side Chains--premise to this approach is simple: if the amino acid side-chain involved in the catalytic activity is chemically modified, the enzyme will be inactivated -identity of modified side chain must be established by standard structural techniques, e.g., isolating and sequencing a modified peptide -problems: chemical modification techniques pose problems both of design and of interpretation -requirements: modification rxn must be specific--only one type of side chain is modified by the reagent (side rxns are minimal) 94 -usually is a problem because many amino acids are nucleophilic so that an electrophile will react with any number of side chains, e.g., those of Cys, Lys, His, Tyr, etc. (a) Applications (i) Cysteines--Hg has a very high affinity for sulphur so mercurial reagents should bring about specific modification of Cys side chains in enzymes (ii) Tyrosine--activated aromatic ring of Tyr side chain is susceptible to electrophilic substitution by I2 or by tetranitromethane (iii) Acylating Reagents--e.g., iodoacetamide or iodoacetate: Cys>Tyr>His>Lys (iv) Arylating Reagents--e.g., 2,4,6-trinitrobenzene sulphonate, 1-fluoro-2,4-dinitrobenzene: Cys>Lys>Tyr>His -the reactivity of any modification rxn should be checked by analysis of the modified enzyme, since the reactivity of a side chain can be considerably influenced by its local environment -change in the selectivity of certain reagents can be brought about by changing the pH -e.g., pH values above 7.0 then side chain of Cys is extremely reactive toward iodoacetate because there is a significant proportion of the ionized form CH2-S-, which is the reactive nucleophile -at pH values below 6, the fraction of the ionized form of the Cys side chain is much less and the side chain of Met is a more 95 reactive nucleophile -by working at pH 5.6 it was possible to react the Met side chain with iodoacetate in isocitrate dehydrogenase from pig heart without any modification of Cys side chains (v) Super-reactive side chains--sometimes one particular amino acid in an enzyme is especially reactive because of its unique environment -e.g., diisopropylfluorophoshate modifies only the side chain of Ser 195 and does not react with any of the other 27 Ser side chains in chymotrypsin or with free Ser -e.g., Lys 126 from glutamate dehydrogenase (beef liver) is especially reactive towards 2,4,6-trinitrobenzenesulphonate (total of 30 Lys in protein) -in both cases above the super-reactive groups were involved in the catalytic mechanisms but this is not always the case (vi) Affinity labelling--to use a modifying reagent which resembles the substrate and will direct the reagent to the active site where it will then react with an amino acid in the vicinity of that site -e.g., bromohydroxyacetone phosphate acts as an affinity label for triosephosphate isomerase -the affinity label binds to the enzyme's active site and the labile Br atom (activated by the adjacent carbonyl group) can be displaced by a suitably positioned nucleophilic amino acid side chain 96 -suicide substrates--a substrate that when acted upon by an appropriate enzyme is converted to product that essentially irreversibly inactivates the enzyme, usually by covalent modification (vii) Interpretation of chemical modification experiments--considerable caution is required because modification of a particular amino acid side chain which leads to inactivation of an enzyme does not necessarily mean that the side chain is directly involved in the catalytic mechanism -the side chain may be somewhat distant from the active site but that modification causes a conformational change in the enzyme leading to loss of activity Criteria for establishment of side chain involvement in catalysis (i) there must be a stoichiometric relationship between the extent of inactivation and the extent of modification so that complete inactivation corresponds to the modification of one side chain per active site (ii) the addition of substrate or competitive inhibitor must protect against inactivation since the side chain is no longer accessible to the modifying reagent (2) Enzyme Engineering and Design (a) Site-directed mutagenesis--recombinant DNA technology has led to the possibility of introducing amino acid replacements at specified positions in a protein provided: 97 (a) gene coding for the protein is available (b) suitable vector for expression of gene exists -site-directed mutagenesis is a powerful method for assessing the importance of particular amino acid side chains in an enzyme -control experiments necessary to determine that the mutation has not altered the overall structure of the enzyme which may have nothing to do with the catalytic mechanism occurring within the active site of the enzyme -techniques such as fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry can be used to assess secondary structure integrity -X-ray crystallography of the mutant proteins is also highly desirable but extremely laborious and time-consuming (b) Insertion/Deletion. -add an extra amino acid into the enzyme or remove/delete one - add or delete a peptide segment -add affinity tags to aid detection or purification (c) Fusion Proteins. -can fuse the genes from two different proteins/enzymes to form a hybrid enzyme that may boast the best attributes/features of both enzymes (d) Novel genes. -synthesize or transfect novel genes into a cell system or organism -introduce normal enzymes into defective systems for therapeutics 98 (e) Non-Natural Amino Acid Mutagenesis (NNAAM) (Nowak, M.W. et al., Science 268, 439, 1995) • used to incorporate an amino acid residue that has novel chemistry not found in the naturally occurring amino acids Applications 1. Incorporation of fluorescent reporter groups -- protein dynamics, conformation, interactions, etc. 2. Positioning of photo-reactive groups for capturing events, trapping conformations, etc. 3. Providing new chemistry for enzyme reactions to enhance rates or provide for new reactions (a) incorporate stronger nucleophiles, electrophiles, more hydrophobes including more heterocyclic side chains, various acid and base groups, stronger redox groups • any specific chemistry that is desired can be incorporated into the enzyme at the specific site that it is needed (b) however, must not be too reactive or unstable but capable of being activated by the appropriate stimulus or chemistry Method 1. At the protein site (amino acid residue) of interest a stop codon is incorporated by mutagenesis into the gene 99 2. The mutated DNA (gene) transcription/translation system is expressed in an in vitro • normally results in a truncated protein 3. If a tRNA bearing an anticodon for the amber stop (a suppressor tRNA) is added to the system then the amino acid appended to this suppressor can be "site-specifically" incorporated the protein • only if the tRNA-NNAA is tolerated by the translation machinery • suppressor tRNA must not be recognized by any of the normal tRNA synthetases in the translation system -- so the NNAA is not removed or replaced by a natural amino acid 4. The tRNA-NNAA must be chemically synthesized • tRNA from yeastPhe with specific base changes is well suited for the E. coli or rabbit reticulocyte lysate translation systems (f) Disulfide bonds. -decrease the number of unfolded conformations by introducing novel disulfide bridges which will increase the stability of the newly engineered enzyme -the longer the loop between the 2 Cys residues the more restricted is the unfolded polypeptide -must be introduced in the proper location or else strain will be present in the new structure which will decrease the stability of the protein's structure 100 101