ENZYMATIC TRANSITION STATES AND TRANSITION STATE ANALOG DESIGN

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P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26QC: ARKAnnual ReviewsAR057-22Annu. Rev. Biochem. 1998. 67:693–720c 1998 by Annual Reviews. All rights reservedCopyright ENZYMATIC TRANSITIONSTATES AND TRANSITIONSTATE ANALOG DESIGNVern L. SchrammDepartment of Biochemistry, Albert Einstein College of Medicine of YeshivaUniversity, Bronx, New York 10461; email: vern@aecom.yu.eduKEY WORDS:catalysis, isotope effects, inhibitor, enzymes, inhibitor designABSTRACTAll chemical transformations pass through an unstable structure called the transition state, which is poised between the chemical structures of the substratesand products. The transition states for chemical reactions are proposed to havelifetimes near 10 13 sec, the time for a single bond vibration. No physical or spectroscopic method is available to directly observe the structure of the transition statefor enzymatic reactions. Yet transition state structure is central to understandingcatalysis, because enzymes function by lowering activation energy. An acceptedview of enzymatic catalysis is tight binding to the unstable transition state structure. Transition state mimics bind tightly to enzymes by capturing a fraction ofthe binding energy for the transition state species. The identification of numeroustransition state inhibitors supports the transition state stabilization hypothesis forenzymatic catalysis. Advances in methods for measuring and interpreting kineticisotope effects and advances in computational chemistry have provided an experimental route to understand transition state structure. Systematic analysis ofintrinsic kinetic isotope effects provides geometric and electronic structure forenzyme-bound transition states. This information has been used to compare transition states for chemical and enzymatic reactions; determine whether enzymaticactivators alter transition state structure; design transition state inhibitors; andprovide the basis for predicting the affinity of enzymatic inhibitors. Enzymatictransition states provide an understanding of catalysis and permit the design oftransition state inhibitors. This article reviews transition state theory for enzymatic reactions. Selected examples of enzymatic transition states are comparedto the respective transition state inhibitors.6930066-4154/98/0701-0693 08.00

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26694QC: ARKAnnual ReviewsAR057-22SCHRAMMCONTENTSINTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694TRANSITION STATE THEORY FOR ENZYME-CATALYZED REACTIONS . . . . . . . . . .Nature of the Transition State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Induced Protein Conformational Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Tight Binding of the Transition State Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Relaxation of the Transition State Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .EXPERIMENTAL APPROACHES TO ENZYMATIC TRANSITIONSTATE STRUCTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Chemical Precedent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Transition State Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Kinetic Isotope Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .EXAMPLES OF ENZYMATIC TRANSITION STATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AMP Nucleosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .S-Adenosylmethionine Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AMP Deaminase and Adenosine Deaminase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Nucleoside Hydrolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Purine Nucleoside Phosphorylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Orotate Phosphoribosyl Transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .NAD and the ADP-Ribosylating Toxins: Cholera, Diphtheria, and Pertussis . . . . . . . . .INHIBITOR PREDICTION FROM TRANSITION STATE INHIBITORS . . . . . . . . . . . . . . .Similarity Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Prediction of Inhibitory Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12717717717718INTRODUCTIONEnzymes catalyze chemical reactions at rates that are astounding relative touncatalyzed chemistry at the same conditions. Typical enzymatic rate enhancements are 1010 to 1015, accomplishing in 1 sec that which would require 300to 30,000,000 years in the absence of enzymes (1). Each catalytic event requires a minimum of three or often more steps, all of which occur within thefew milliseconds that characterize typical enzymatic reactions. According totransition state theory, the smallest fraction of the catalytic cycle is spent inthe most important step, that of the transition state. However, this step cannotoccur without participation of the enzymatic forces that occur as the Michaeliscomplex is transformed into the transition state by precise alignment of catalyticgroups by enzymatic and substrate conformational changes (Figure 1).The original proposals of absolute reaction rate theory for chemical reactions defined the transition state as a distinct species in the reaction coordinatethat determined the absolute reaction rate (2). Soon thereafter, Linus Paulingproposed that the powerful catalytic action of enzymes could be explained byspecific tight binding to the transition state species (3). Because reaction rateis proportional to the fraction of the reactant in the transition state complex,the enzyme was proposed to increase the concentration of the reactive species.This proposal was formalized by Wolfenden and coworkers, who hypothesizedthat the rate increase imposed by enzymes is proportional to the affinity of

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26QC: ARKAnnual ReviewsAR057-22ENZYMATIC TRANSITION STATES695Figure 1 Reaction coordinate diagram for conversion of substrate (A) to enzyme-bound products(EP). Symbols are R H-bond acceptor, H H-bond donor, and are ionic charges, and represents hydrophobic sites. The solid line is an example of fully rate-limiting transition stateformation, providing intrinsic kinetic isotope effects. The energetic barriers (dashed lines) labeled“forward commitment” and “reverse commitment” make substrate binding and/or product releaserate limiting and suppress kinetic isotope effects. The transition state has unique properties of chargeand optimal H-bond alignment not found in any of the reactant species. In this example, chargerepulsion serves to clear the catalytic site and restore the enzyme to the open form (E) after k5.the enzyme for the transition state structure relative to the Michaelis complex(4; Figure 2). Because enzymes typically increase the noncatalyzed reactionrate by factors of 1010–1015, and Michaelis complexes often have dissociationconstants in the range of 10 3–10 6 M, it is proposed that transition state complexes are bound with dissociation constants in the range of 10 14–10 23 M.Analogs that resemble the transition state structures should therefore providethe most powerful noncovalent inhibitors known, even if only a small fractionof the transition state energy is captured.Reviews from 1976, 1988, and 1995 listed enzymes known to interact withtransition state inhibitors, defined as tight binding inhibitors that resemble thehypothetical transition states or intermediates for various enzymes (5–7). Between 1988 and 1995, the list grew from 33 to 132 enzymes. In 1976 and 1988,most of the inhibitors were natural products. The 1995 list of enzymes andtransition state inhibitors is dominated by intentionally synthesized inhibitors

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26696QC: ARKAnnual ReviewsAR057-22SCHRAMMFigure 2 The thermodynamic box that predicts transition state binding affinity (upper left) compares the rates of uncatalyzed (kuncat) and enzymatic (kenz) reactions and assumes that the transmission coefficients from [A]‡ and [EA]‡ are equal. The dissociation constants for EA and [EA]‡ are‡given by Kd and Kd . Transition state inhibitors (lower left) invoke the unique ionization and conformational structure found exclusively in the transition state (see Figure 1). Catalysis is preventedby a stable bond at the reaction center (represented by the solid dot). The energetics of catalysisand binding an energetically perfect transition state analog are compared in the reaction or bindingcoordinate diagram. Energy of enzymatic transition state stabilization (11G‡ ) is converted intobinding energy for the transition state inhibitor (11G-I‡ ) to form the stable EI‡ complex, whichcannot escape by the product release pathway. The slow-onset inhibition common with transitionstate inhibitors occurs after formation of a readily reversible E I‡ complex. The rate of onset for‡‡tight-binding inhibitors is k2 and the rate of escape is k 2 . Note the unfavorable energetic barrier‡for escape from the stable EI complex.in response to inhibitor development for the AIDS protease, β-lactamases,metalloproteinases, cyclooxygenases, and a growing list of enzymes that aretargets for pharmaceutical intervention. Because many inhibitor developmentsare proprietary, the list for 1995 is likely to be an incomplete representation ofthe known list of transition state inhibitors.In 1947, an experimental approach to chemical transition states was discovered by Bigeleisen and coworkers, who, along with others, established therelationships among isotopic substitution, altered reaction rates, and alteredbonding between reactants and transition states (8–10). A few applications

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26QC: ARKAnnual ReviewsAR057-22ENZYMATIC TRANSITION STATES697of individual kinetic isotope effects were made to enzymatic reaction mechanisms before 1970. Qualitative results from these studies indicated whether thebond changes to the isotopically labeled atom occurred in the rate-limitingstep (e.g. Figure 1). The Steenbock Symposium of 1976, “Isotope Effectson Enzyme-Catalyzed Reactions,” provided the impetus for additional studieson enzymes (11). The 1978 book Transition States of Biochemical Processesincluded the provocative and fundamentalist position of RL Schowen that “theentire and sole source of catalytic power is the stabilization of the transitionstate; that reactant-state interactions are by nature inhibitory and only wastecatalytic power” (12, p. 78). These works bridged the gap between chemicaland biological transition states, recognizing that even complex biological transformations involve formation of one or more defined transition states and aretherefore susceptible to transition state analysis based on the measurement ofkinetic isotope effects. Development of steady state and pre–steady state kineticmethods to reveal intrinsic isotope effects permitted interpretation of results interms of transition state structure (13–17).Although the focus of this review is enzymatic transition states and relatedinhibitors, transition state similarity is not necessary for tight-binding inhibition of enzymes. Any combination of multiple favorable hydrogen, ionic, orhydrophobic bonds between enzyme and substrate can provide the summationof binding interactions leading to tight-binding inhibition and is the basis forinhibitory screening from chemical and combinatorial libraries. Examples ofsuch inhibitors are found in nature as antibiotics. Streptomycin, erythromycin,and rifampicin are examples of complex natural products that have no knownsimilarity to the transition states involved in protein and RNA synthesis. Inhibitors designed to match contacts in the catalytic site or those that are stable analogs of the substrate can bind tightly but do not qualify as transitionstate inhibitors. One example is the tight binding of methotrexate to dihydrofolate reductase, in which the analog is bound upside down with respect to substrate in the catalytic site (18). Another example is 9-deaza-9-phenyl-guanine,a powerful inhibitor of purine nucleoside phosphorylase. It was designed fromthe X-ray crystal structure of substrate and product complexes and does notresemble the transition state (19, 20) (Figure 3).TRANSITION STATE THEORY FORENZYME-CATALYZED REACTIONSNature of the Transition StateAs substrate progresses from the Michaelis complex to product, chemistryoccurs by enzyme-induced changes in electron distribution in the substrate. Enzymes alter the electronic structure by protonation, proton abstraction, electron

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26698QC: ARKAnnual ReviewsAR057-22SCHRAMMFigure 3 Tight-binding inhibitors of purine nucleoside phosphorylase and dihydrofolate reductasethat are not transition state analogs despite high-affinity binding. The Km values for substrates andequilibrium Ki values for the inhibitors are shown.transfer, geometric distortion, hydrophobic partitioning, and interaction withLewis acids and bases. These are accomplished by sequential protein and substrate conformational changes (Figure 1). When a constellation of individuallyweak forces are brought to bear on the substrate, the summation of the individual energies results in large forces capable of relocating bonding electrons tocause bond-breaking and bond-making. Substrates of modest molecular size(for example, glucose), bound in the active sites of proteins, typically interact with two H-bonds at each donor/acceptor site. These interactions alone, intypical H-bond energies of 3 kcal/mol/H-bond, can provide over 30 kcal/mol ofenergy toward redistribution of electrons and therefore toward ionizations andcovalent bond changes. The restricted geometry and hydrophobic environmentallow short H-bonds to form that would be unfavorable in solution (21, 22).The lifetime for chemical transition states is short, approximately 10 13 sec,the time for conversion of a bond vibrational mode to a translational mode(23, 24). This theory requires reexamination for enzymes, because the proteindomain motion resulting in transition state formation may stabilize the alteredbond lengths of the bound transition state for a lifetime sufficient for 101–106

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26QC: ARKAnnual ReviewsAR057-22ENZYMATIC TRANSITION STATES699vibrations. This hypothesis is indicated by the dimpled transition state featurelabeled with a question mark in Figure 1; it remains unexplored except by computational theory. Enzymes that form transient covalent intermediates requiretwo distinct transition states. These transition states are surrounded by lowerenergy complexes and should be distinguished from the altered vibrational stateimplied by the question mark in Figure 1. The classic interpretation of the transition state supposes a short lifetime during which an infinitesimal force towardproduct or substrate leads to EP0 and EA0 respectively.Induced Protein Conformational ChangeThe energetic problem that must be solved by enzymes is to bind tightly onlyto the unstable transition state structure while avoiding tight binding to the substrate and products. Enzymes often bind to the substrate at diffusion-controlledrates, and subsequent conformational or electronic changes are mandatory forcatalysis. Placing the enzyme-bound substrate in the solvent-restricted environment of the closed catalytic site permits the subsequent events to occur inthe altered solvent of the catalytic site.Presenting the enzyme with a transition state mimic results in a mismatch ofthe substrate-recognizing features. Many transition state inhibitors are slowonset, typified by a rapid weak binding followed by a slow tight-binding interaction. The energetics of this interaction (Figure 2) show rapid formation ofthe encounter complex E I‡ (similar to the EA complex of Figure 1) followedby a difficult (high-energy, slow) entry to the stable EI‡ complex. The boundinhibitor does not induce the conformational change with the efficiency of substrate and requires time to permit the transition state conformational change tooccur on the protein. This time corresponds to the slow onset of tight-bindinginhibition commonly observed with transition state mimics (6). These dataargue that the substrate actively induces the catalytic conformational change,because the rate of catalysis is substantially greater than the rate at which mosttight-binding inhibitors induce enzymes into the transition state configuration.This view of transition state inhibitor interaction predicts that near-perfect inhibitors would still exhibit slow-onset inhibition because the enzyme is designedto recognize the ground state of the substrate. However, some tightly boundinhibitors are reported to achieve inhibition on the time scale of catalysis. Forexample, the inhibition of adenosine deaminases by purine riboside and of cytidine deaminase by pyrimidin-2-one riboside is fast (25–26). These inhibitorsare estimated to bind with dissociation constants of 10 13 and 10 12 M respectively, approaching the hypothetical 10 16 M dissociation constant for the actualtransition states. Rapid onset occurs because these are half-reaction substratesfor the enzymes. The deaminases contain a tightly bound zinc that acts as thecatalytic site base to ionize a water molecule to the hydroxide and position it near

P1: DPI/aryP2: RPK/ARK/plbMay 12, 199814:26700QC: ARKAnnual ReviewsAR057-22SCHRAMMFigure 4 Hydration of purine riboside by a catalytic site hydroxyl generated at the tightly boundZn2 . The hydrated purine is bound tightly. An analogous reaction occurs with cytidine deaminase.the reactive carbon of the aromatic rings (27; Figure 4). Enzymatic protonationof the adjacent aromatic ring nitrogen assists in the required rehybridization ofthe rings. These inhibitors take advantage of the substrate-induced transitionstate configuration to cause rapid hydration and tight binding of the hydratedspecies. The sp3 hybridization at the reactive carbon is a transition state feature,and without the amino leaving group, a stable complex is formed.Tight Binding of the Transition State StructureBinding energies of enzymatic transition states are generated by the realignment of substrate (Michaelis) contacts as the enzyme and substrate mutuallychange their structures toward the transition state (Figure 1). The strong dependence of hydrogen and ionic bond energy on bond distance, angle, solventenvironment, and relative pKa values can be invoked to explain the increases inbinding forces of the transition state complex relative to the Michaelis complex(21, 28). Structural rearrangements tighten the protein around the catalytic siteto exclude solvent and to make stronger electrostatic contacts. These are shownas well-aligned H-bonds at the transition state and as ionic attraction and repulsion as catalytic forces (Figure 1). Enzymatic reactions usually demonstratedistinct pKa values for substrate binding and kcat, testifying to the ionic changesbetween the enzyme substrate and enzyme transition state complexes (29, 30).A common mechanism for enzymatic catalysis is to generate differential chargebetween substrate and transi

Figure 1 Reaction coordinate diagram for conversion of substrate (A) to enzyme-bound products (EP). Symbols are R DH-bond acceptor, H DH-bond donor,Cand ¡are ionic charges, and represents hydrophobic sites. The solid line is an example of fully rate-limiting transition state formation, providing intrinsic kinetic isotope effects.

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