Key Concepts.

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1 Lectures 13-14: Enzyme Catalytic Mechanisms [PDF] Reading: Berg, Tymoczko & Stryer, Chapter 9, pp Updated on: 2/7/07 at 9:15 pm movie of chemical mechanism of serine proteases (from Voet & Voet, Biochemistry, 3rd edition, 2004, Wiley) Key Concepts Mechanisms used by enzymes to enhance reaction rates include: (1st 4 mechanisms based on BINDING of substrate and/or transition state) Proximity & orientation Desolvation (one type of electrostatic catalysis) Preferential binding of the transition state Induced fit General acid/base catalysis Covalent (nucleophilic) catalysis Metal ion catalysis (Electrostatic catalysis) The chemical mechanism of serine proteases like chymotrypsin illustrates not only proximity and orientation, but also: Transition state stabilization. Covalent catalysis, involving a catalytic triad of Asp, His and Ser in the active site. general acid-base catalysis electrostatic catalysis Learning Objectives Discuss (briefly explain): 8 general catalytic mechanisms used by enzymes to increase the rates of chemical reactions. (You won't be asked on an exam to simply LIST them, but you could be expected to explain any one -- or 2 or 3 or 4 -- of them.) Explain why peptide bonds are kinetically stable in the absence of a catalyst, given that equilibrium lies far in the direction of hydrolysis in 55.5 M H 2 O. (Why is any specific reaction a slow reaction?) Describe the chemical mechanism of hydrolysis of specific peptide bonds in chymotrypsin, including the following: What is the "job" of the catalyst (the protease), i.e., what group needs to be made more susceptible to nucleophilic attack? Describe substrate binding, including the role and chemical nature of the "specificity pocket" in chymotrypsin, and which peptide bond in the substrate (relative to the specificity group) will be cleaved. Draw the structure of the catalytic triad at the beginning of the reaction, and explain how the states of ionization and hydrogen bonding pattern of those 3 groups change step by step during catalysis.. Explain the role of each member of the catalytic triad in the reaction. Identify the nucleophile that attacks the carbonyl carbon in acylation; identify the nucleophile that attacks the carbonyl carbon in deacylation. What is an acyl group? Describe the acyl-enzyme intermediate, including identifying the type of bond attaching the acyl group to the enzyme (Is it an amide linkage? anhydride? ester? etc.) and how that acyl group relates to the structure of the original substrate. Draw the structures of each of the tetrahedral intermediates in the reaction.. Identify the leaving group coming from each of the tetrahedral intermediates as the intermediate breaks down. State what is being acylated and deacylated in the chymotrypsin reaction (be specific about the functional group involved). Explain the role of the "oxyanion hole" in the mechanism. Describe which type(s) of general catalytic mechanisms (first learning objective above) are used by chymotrypsin, and how. Compare (very briefly, just the bottom line ) the overall 3-dimensional structures of chymotrypsin, trypsin, and elastase, and compare the substrate binding specificities of those 3 enzymes, explaining the relationship of the specificity site/pocket structure to the differences in substrate specificity.. How do 3 other classes of proteases (besides the serine proteases) generate nucleophiles potent enough to attack a peptide carbonyl group? To which protease class does HIV protease belong? Describe the quaternary structure and symmetry of the HIV Page 1 of 14

2 protease and where in the quaternary structure the active site residues are located. GENERAL MECHANISMS IN ENZYME CATALYSIS Different enzymes use different COMBINATIONS of mechanisms to reduce activation energy and thus increase rate of reaction. 7 "types" of mechanisms below are really "overlapping" concepts in many cases. first 4 related to BINDING of substrate and/or transition state, so reaction takes place in active site, not in bulk solution 1. PROXIMITY AND ORIENTATION (catalysis by approximation) Proximity: Reaction between bound molecules doesn't require an improbable collision of 2 molecules. They're already in "contact" (increases local concentration of reactants). Orientation: Reactants are not only near each other on enzyme, they're oriented in optimal position to react. The improbability of colliding in correct orientation is taken care of. 2. DESOLVATION -- active site gets reactants out of H 2 O Lower dielectric constant environment than H 2 O (i.e. more nonpolar environment), so stronger electrostatic interactions (strength is inversely related to dielectric constant) Reactive groups of reactants are protected from H 2 O, so H 2 O doesn't compete with reactants. (H 2 O won't react to give unwanted byproducts, e.g., by hydrolysis of some reactive intermediate in the reaction that was supposed to transfer its reactive group to another substrate.) 3. TIGHT TRANSITION STATE BINDING (used to be called "strain and distortion") Free energy of transition state (peak of free energy barrier on reaction diagram) is lowered because its "distortion" (electrostatic or structural) is "paid for" by tighter binding of transition state than of substrate. 4. INDUCED FIT Conformational change resulting from substrate binding Binding may stabilize a different conformation of either enzyme or substrate or both Conformational change orients catalytic groups on enzyme promotes tighter transition state binding, and/or excludes H 2 O (obviously related to tight transition state binding, too ) e.g., hexokinase binding of glucose (1st reaction in glycolysis) Lehninger Biochemistry (Nelson & Cox) Fig. 8-21: Induced fit in hexokinase. Ends of the U-shaped enzyme hexokinase pinch toward each other -- conformational change induced by binding D-glucose (red) Jmol routine -- hexokinase conformational change Slide your computer mouse across the structure below in the html file to see the conformational rearrangement when glucose binds. Note the substantial structural change in hexokinase between the free enzyme and the glucose complex: Page 2 of 14

3 NOTE: 1st 4 concepts (above) in catalysis are rather general. all related to BINDING of substrate and/or transition state Reaction takes place in active site, not in bulk solution. Other catalytic mechanisms (below) involve specific groups and chemical mechanisms that depend on the specific reaction. 5. GENERAL ACID-BASE CATALYSIS Specific functional groups in enzyme structure are positioned to donate a proton (act as a general acid), or accept a proton (act as a general base). helps enzyme to avoid unstable charged intermediates in reaction Conjugate acid form of group that donates a proton (acts as a general acid) in catalysis and thus is converted into conjugate base form has to then accept a proton (act as a general base) later in catalytic mechanism for catalyst to be regenerated in its original conjugate acid form. (Converse also true -- group acting first as general base to accept a proton, must later in catalytic cycle donate that proton to be regenerated as a catalyst ready for another catalytic cycle.) examples of general acid/base catalysts among protein functional groups: His imidazole α-amino group thiol of Cys R group carboxyls of Glu, Asp ε-amino group of Lys aromatic OH of Tyr guanidino group of Arg 6. COVALENT CATALYSIS rate acceleration by transient formation of a COVALENT catalyst-substrate bond covalent intermediate is more reactive in next step in reaction, so that step has lower activation energy than it would have for a non-covalent catalytic mechanism -- enzyme alters pathway to get to product. Nucleophile: an electron-rich group that attacks nuclei examples of nucleophiles among protein functional groups: unprotonated His imidazole unprotonated α-amino group unprotonated ε-amino group of Lys unprotonated thiol (thiolate anion, -S - ) of Cys aliphatic -OH of Ser R group carboxylates of Glu, Asp Also some coenzymes, e.g., thiamine pyrophosphate (TPP) & pyridoxal phosphate (PLP) 7. METAL ION CATALYSIS (several catalytic roles) Metal ions can be either tightly bound (metalloenzymes), i.e., as a prosthetic group (usually transition metal ions, e.g., Fe 2+ or Fe 3+, Zn 2+, Cu 2+, Mn 2+...) OR loosely bound, binding reversibly and dissociating from enzyme (usually Na +, K +, Mg 2+, Ca 2+...) Functions of metal ions in catalysis include: A. Binding and orientation of substrate (ionic interactions with negatively charged substrate) B. Redox reactions (e.g. Fe 2+ / Fe 3+ in some enzymes) C. Shielding or stabilizing negative charges on substrate or on transition state (electrophilic catalysis) example: Enzymes that bind ATP (adenosine triphosphate) require Mg 2+ to be bound to the nucleotide (so ligand is actually Mg 2+ ATP) in order to Page 3 of 14

4 shield negative charges, and orient the ATP substrate e.g., Kinases: enzymes catalyzing phosphoryl transfer involving ATP or other nucleoside triphosphates All kinases require Mg 2+ for activity, but it's actually in complex with nucleotide (usually ATP). example: hexokinase (catalyzes transfer of the terminal phosphate group from ATP to glucose, producing glucose-6- phosphate and ADP as products) [8. ELECTROSTATIC EFFECTS] concept not always "listed" separately because it s involved in many other aspects of catalytic mechanisms Some examples: providing lower dielectric constant of environment in active site (hydrophobic environment) altering pk values of specific functional groups stabilizing a particular conformation of critical groups in active site by electrostatic interactions stabilizing (binding) a charged intermediate or transition state by providing an oppositely charged enzyme group nearby. Enzyme Chemical Mechanisms (Chymotrypsin as an example) Chymotrypsin: Berg, Tymoczko & Stryer, 6th ed. Fig. 9.6: 3-dimensional structure of chymotrypsin, showing active site residues digestive protease synthesized in mammalian pancreas and secreted in inactive form as a single polypeptide chain (chymotrypsinogen) activated by proteolytic processing to rearrange conformation to active enzyme, which because of the activating peptide bond "clips" has 3 chains. structure stabilized by disulfide bonds (true for many extracellular proteins) PROTEASES: Reaction catalyzed = hydrolysis of peptide bonds in vivo, function in digestion of nutrient protein, and in protein turnover (degradation of proteins that are old or no longer needed as conditions change) Peptide bond hydrolysis (S N 2 attack by :O of water on the carbonyl C of the peptide bond) Equilibrium (in 55.5 M H 2 O) lies FAR to the right, but in absence of catalyst, reaction is extremely slow (fortunately -- or our bodies would all be puddles of amino acids in solution!) Peptide bonds "kinetically stable" Mechanism of uncatalyzed reaction: simple nucleophilic attack by :O of H 2 O on carbonyl C of peptide bond, forming tetrahedral intermediate Tetrahedral intermediate then breaks down as the amine "half" of original peptide leaves. Reason uncatalyzed reaction is so slow: partial double bond character of peptide bond makes carbonyl carbon much less reactive than carbonyl carbons in carboxylate esters. Page 4 of 14

5 Catalytic task of proteases is to make that normally unreactive carbonyl group more susceptible to nucleophilic attack by H 2 O. 4 classes of proteases based on different mechanisms to enhance the susceptibility of the carbonyl group to nucleophilic attack 1. Serine proteases (e.g. chymotrypsin) -- covalent catalysis, with initial nucleophilic attack carried out by an enzyme Ser-O(H) group made into a potent nucleophile with assistance of nearby His imidazole that acts as a general base 2. Cys proteases -- again, covalent catalysis, with initial nucleophilic attack carried out by an enzyme Cys-S(H) group made into a potent nucleophile with assistance of nearby His imidazole that acts as a general base 3. Asp proteases -- nucleophile is HOH itself, assisted by 2 Asp residues, general base catalysis by 1st Asp carboxyl group and orientation/polarization of substrate carbonyl by 2nd Asp residue 4. Metalloproteases -- again, nucleophile is HOH, but assisted by binding to a metal (e.g. Zn 2+ ) and by general base catalysis by some enzyme base group, e.g. Glu-COO -. A detailed look at a SERINE PROTEASE, CHYMOTRYPSIN Chymotrypsin makes carbonyl C of peptide bond more reactive by changing pathway of reaction. Covalent catalysis by a Ser residue, with assistance of a general base (His) Overall reaction on enzyme occurs in 2 separate "half reactions" (2 "phases" of catalysis), with a metastable covalent intermediate ("acyl-enzyme intermediate") between the 2 half reactions. OVERVIEW OF CHYMOTRYPSIN MECHANISM, 2 HALF REACTIONS: First step/phase ("acylation") Enzyme provides potent nucleophile, a specific Ser OH group. Ser OH made more nucleophilic than usual with assistance of nearby His residue as general base Nucleophilic attack on substrate --> covalent intermediate, the acyl enzyme intermediate (actually a carboxylate ester of carboxylate "half" of original substrate, attached to enzyme's Ser R group that's the alcohol component of the ester). Amine "half" of original peptide/protein released as product (P 1 ) at end of first phase. Second phase ("deacylation") 2nd substrate, H 2 O, is nucleophile, attacking carbonyl C of the carboxylate ester of acyl enzyme, again with assistance of active site His residue as general base. Ester bond of intermediate is hydrolyzed to regenerate alcohol component (the enzyme chymotrypsin, with its Ser-OH free again) and carboxylic acid component, the 2nd product (P 2 ) (carboxyl "half" of original substrate peptide/protein). Berg, Tymoczko & Stryer, 6th ed. Fig. 9.5: Covalent catalysis (2 phases of chymotrypsin-catalyzed peptide bond hydrolysis) OVERALL MECHANISM OF CHYMOTRYPSIN 2 chemical "phases" S = polypeptide; P 1 = AMINE product (2nd "half" of substrate) R 2 -NH 2 E P 2 = COVALENT intermediate, the ACYL-ENZYME intermediate P 2 = CARBOXYLATE product (1st "half" of substrate) R 1 -COOH Page 5 of 14

6 SUMMARY: 2-phase process -- overall chemical steps in the 2nd phase are almost an exact repeat of the processes in the first phase. THE CATALYTIC TRIAD 3 amino acid residues in active site in a hydrogen-bonded network: Ser (residue #195) His (residue #57) Asp (residue #102) essential for effective catalytic activity in chymotrypsin converts OH group of Ser 195 into a potent nucleophile Berg, Tymoczko & Stryer Fig. 9.7: The catalytic triad of chymotrypsin. movie of chemical mechanism of serine proteases Berg, Tymoczko & Stryer, 6th ed. Fig. 9.8: Chemical Mechanism of Chymotrypsin -- whole summary figure FIRST PHASE OF CATALYSIS (PHASE I, ACYLATION): Page 6 of 14

7 Formation of acyl-enzyme covalent intermediate and generation of P 1, the amine product 1. FORMATION OF ES COMPLEX enzyme binds substrate (peptide or protein), with aromatic bulky hydrophobic side chain "specificity group" in "pocket" bound susbstrate is positioned for the peptide bond on carbonyl side (i.e., "carboxyl" side) of that residue to be cleaved. Polypeptide chain of substrate also forms a short β- sheet (hydrogen bonds) with a β strand of enzyme in binding site. 2. FORMATION OF FIRST TETRAHEDRAL INTERMEDIATE Oxygen atom of active site Ser-OH is activated by hydrogen bond network linkage to His (imidazole ring N:) in catalytic triad His acts as proton acceptor (general base catalysis), taking proton from Ser-OH to become HisH + O atom becomes a potent nucleophile, to attack the carbonyl C of the peptide bond to be cleaved. Ser-O( - ) (potent nucleophile) carries out nucleophilic attack on carbonyl C of substrate (nucleophilic catalysis, i.e. covalent catalysis) --> COVALENT bond to carbonyl C (1st tetrahedral intermediate). [Proton transfer and nucleophilic attack may be concerted (occur at same time), so Ser-O may not actually exist as an oxyanion/alkoxide ion (-O - )]. Asp in catalytic triad a) helps maintain the perfect orientation of His and Ser residues in the hydrogen bonded network, and b) facilitates the H + transfer by electrostatic stabilization of the HisH + after it has accepted the proton. Product of this step = FIRST TETRAHEDRAL INTERMEDIATE structure presumed similar to that of TRANSITION STATE, with negatively charged "carbonyl" OXYGEN (not a carbonyl group anymore), an OXYANION. TRANSITION STATE STABILIZATION Berg, Tymoczko & Stryer, 6th ed. Fig. 9.9: The "oxyanion hole", an area in the active site of serine proteases that binds the transition state particularly tightly. Page 7 of 14

8 Active site binds oxyanion more tightly than it bound original carbonyl group of the substrate. Another hydrogen bond forms between tetrahedral oxyanion and enzyme groups around it (which form the "oxyanion hole" portion of the active site) That hydrogen bond couldn't form to carbonyl form of the oxygen (=O) but can form now because of structural change (lengthening of C-O bond) on forming tetrahedral intermediate. Also, hydrogen bonds to negatively charged oxygen are stronger than to neutral O. 3. FORMATION OF ACYL-ENZYME INTERMEDIATE FIRST tetrahedral intermediate BREAKS DOWN (original amide (peptide) bond CLEAVES) HisH + donates a proton to the amino "half" of the original substrate (HisH + acts as a general acid) to generate R 2 -NH 2. Breaking of amide bond (departure of amine product, P 1 ) --> conversion of oxyanion back into a C=O, still covalently attached to the Ser residue of the enzyme, forming ACYL-ENZYME INTERMEDIATE. Original carbonyl group of peptide bond is now a carbonyl group again, but it's covalently attached to the Ser-O. Acyl-enzyme has a covalent ESTER linkage between "carboxyl half" of original peptide substrate and O from the Ser alcohol R group. Page 8 of 14

9 Breaking of amide bond (departure of amine product, P 1 ) --> conversion of oxyanion back into a C=O, still covalently attached to the Ser residue of the enzyme, forming ACYL-ENZYME INTERMEDIATE. Original carbonyl group of peptide bond is now a carbonyl group again, but it's covalently attached to the Ser-O. Acyl-enzyme has a covalent ESTER linkage between "carboxyl half" of original peptide substrate and O from the Ser alcohol R group. 4. AMINE PRODUCT (R 2 -NH 2 ) DISSOCIATES FROM ACTIVE SITE, i.e. P 1 LEAVES. SECOND PHASE OF CATALYSIS (PHASE II, DEACYLATION) Breakdown of acyl-enzyme covalent intermediate by reaction with H 2 O (HYDROLYSIS) and release of P 2, the carboxylic acid product This phase (second "half reaction") is almost an exact repeat of the first in terms of catalytic steps/mechanisms -- nucleophilic attack facilitated by His acting as general base (but nucleophile is H 2 O), formation of second tetrahedral intermediate with transition state stabilization by binding of C-O - in oxyanion hole, and breakdown of 2nd tetrahedral intermediate (cleavage of what had been the ester bond in acyl-enzyme intermediate, with HisH + as general acid catalyst) to regenerate enzyme Ser-OH and release P 2, the carboxylic acid product (from the original peptide/protein substrate). 5. BINDING OF THE SECOND SUBSTRATE, H 2 O, IN THE ACTIVE SITE. Page 9 of 14

10 6. FORMATION OF THE SECOND TETRAHEDRAL INTERMEDIATE HOH forms hydrogen bond with HisN: (just like Ser-OH did in first phase) in hydrogen bond network. His again acts as a general base, to become HisH +, activating O from H 2 O to make it a potent nucleophile, to attack carbonyl C of acyl-enzyme intermediate (an ester). As His accepts proton from HOH (so His picks up + charge), O of water becomes a potent nucleophile nucleophilic attack on carbonyl C of the acyl-enzyme intermediate (nucleophilic catalysis, i.e. covalent catalysis) COVALENT bond between OH of water and carbonyl C (2nd tetrahedral intermediate). Asp in catalytic triad a) helps maintain the perfect orientation of the hydrogen bonded network, and b) facilitates the H + transfer by electrostatic stabilization of the HisH + after it has accepted the proton. Product of this step = SECOND TETRAHEDRAL INTERMEDIATE structure presumed similar to that of TRANSITION STATE, with negatively charged "carbonyl" OXYGEN (not a carbonyl group anymore), an OXYANION. Note that SECOND tetrahedral intermediate has an -OH group on it (from H 2 O) instead of amido group of amine "half" of original substrate as in FIRST tetrahedral intermediate. TRANSITION STATE STABILIZATION (See Berg, Tymoczko & Stryer, 6th ed. Fig. 9-9 above, the oxyanion hole.) Active site binds oxyanion more tightly than it bound original carbonyl group of the substrate. Another hydrogen bond forms between tetrahedral oxyanion and enzyme groups around it (which form the "oxyanion hole" portion of the active site) That new hydrogen bond couldn't form to carbonyl form of oxygen (=O) but can form now because of structural change (lengthening of C-O bond) on forming tetrahedral intermediate. Also, hydrogen bonds to negatively charged oxygen are stronger than to neutral O. 7. BREAKDOWN OF SECOND TETRAHEDRAL INTERMEDIATE Original ester bond (from acyl-enzyme) CLEAVES. HisH + (general acid) donates proton back to Ser O (generating alcohol product of hydrolysis of acyl-enzyme, Ser-OH Ester bond from acyl-enzyme intermediate breaks --> carboxylic acid product (R 1 -COOH) from original substrate. Page 10 of 14

11 8. Carboxylic acid product dissociates from active site, i.e. P 2 leaves. Enzyme molecule now in its original state, with His imidazole in neutral form, catalytic triad appropriately hydrogen-bonded, and active site ready to bind another molecule of substrate and do it all again. Berg, Tymoczko & Stryer, 6th ed. Fig. 9.10: The hydrophobic "specificity pocket of chymotrypsin (responsible for its substrate specificity). Position of aromatic ring bound in pocket is shown in green in center. movie of chemical mechanism of serine proteases (same chemical mechanism for all the serine proteases, but chymotrypsin is the example shown) Catalytic triads in other hydrolytic enzymes Berg, Tymoczko & Stryer, 6th ed. Fig. 9.12: 3-dimensional folds (tertiary structures) of chymotrypsin (red) and trypsin (blue). Only the backbone tracings of α carbon positions are shown. family of mammalian serine proteases: e.g., chymotrypsin, trypsin & elastase obviously homologous -- primary structures about 40% identical and 3- dimensional folds are nearly identical suggests common evolutionary origin with a single ancestral gene that duplicated a number of times, after which sequences and substrate specificities diverged (example of divergent evolution.) Family also includes many proteolytic enzymes in the blood clotting Page 11 of 14

12 cascade. Berg, Tymoczko & Stryer, 6th ed. Fig. 9.13: The S 1 specificity pockets of chymotrypsin, trypsin, and elastase. Substrate binding sites on enzymes where "R 1 " group of the substrate binds ("R 1 " = R group of the amino acid residue contributing the carbonyl group of the peptide bond to be cleaved). Trypsin cleaves peptide bonds on carbonyl side ("after") long + charged residues (R 1 = Lys + or Arg + ) specificity "assisted" by Asp - residue in bottom of S 1 site. S 1 site of elastase is partly closed off so only small side chains may enter Elastase cleaves after small neutral residues (e.g., Gly and Ala). Subtilisin a bacterial hydrolytic enzyme no apparent evolutionary relationship to mammalian chymotrypsin/trypsin family (no primary structure or tertiary structural resemblance) uses same catalytic mechanism: with a catalytic serine assisted by a His and an Asp residue (catalytic triad), in same orientation example of convergent evolution, independent evolution of same catalytic strategy (The chymotrypsin mechanism must be a very effective hydrolytic mechanism!) Berg, Tymoczko & Stryer, 6th ed. Fig. 9.14: Catalytic triad and oxyanion hole of subtilisin, a bacterial protease Page 12 of 14

13 site-directed mutagenesis studies on structural alterations in subtilisin active site gene for the enzyme was cloned specific mutations in the catalytic triad residues (individually and in combination) introduced by molecular biological methods mutant enzymes expressed, purified and studied. Berg, Tymoczko & Stryer, 6th ed. Fig. 9.16: Site directed mutagenesis of subtilisin. 1st letter is normal (wild type) residue in that (number) position; 2nd letter is mutant amino acid residue replacing normal residue at that position. Results: Mutations in catalytic triad residues have dramatic effects on k cat (the turnover number). NOTE log scale -- mutation of Ser or His reduces k cat by a factor of about 10 6! However, also note that what might seem to be "fatally" modified (mutated) enzymes still have higher k cat values than k cat for uncatalyzed reaction, by factor of ~ Other classes of proteases use other strategies than a serine nucleophile, but they all generate a potent nucleophile to attack the peptide carbonyl group. Cys proteases: nucleophile is a His-activated Cys thiol (general base catalysis by His) Asp proteases: nucleophile is HOH itself assisted by 2 Asp residues, general base catalysis by 1 Asp carboxyl group and orientation/polarization of substrate carbonyl by 2nd Asp residue Metalloproteases: nucleophile is HOH assisted by binding to a metal (e.g. Zn 2+ ) and by general base catalysis by some enzyme base group, e.g. Glu-COO -. Berg, Tymoczko & Stryer, 6th ed. Fig. 9.18: Activation strategies for 3 more classes of proteases (in addition to the Ser proteases) Berg, Tymoczko & Stryer, 6th ed. Figs and 9.21: HIV protease an Asp protease a homodimer Each of the 2 identical subunits contributes an Asp to the active site. Note the 2 catalytic Asp residues, 1 from each subunit, on opposite sides of the 2-fold axis of symmetry (below the bound crixivan in Fig. 9.21). Structure in Fig has substrate binding pocket indicated, with the 2 catalytic Asp residues in ball-and-stick structures. "Flaps" (a portion of each polypeptide chain, labeled) close down after substrate binds (induced fit). structure shown in Fig is in complex with an inhibitor, crixivan, which has a conformation that approximates the 2-fold symmetry of the enzyme. Crixivan thus inhibits HIV protease without affecting normal cellular Asp proteases, which don't have the 2-fold symmetry that HIV protease has. Crixivan was designed to mimic the tetrahedral intermediate (and thus the transition state) -- it's a transition state analog, with Page 13 of 14

14 groups to bind various sub-pockets in substrate binding site. Department of Biochemistry & Molecular Biophysics The University of Arizona Copyright ( ) 2007 All rights reserved. Page 14 of 14