Báo cáo Y học: Barrier passage and protein dynamics in enzymatically catalyzed reactions

Eur. J. Biochem. 269, 3103–3112 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03021.x MINIREVIEW Barrier passage and protein dynamics in enzymatically catalyzed reactions Dimitri Antoniou1, Stavros Caratzoulas1,*, C. Kalyanaraman1, Joshua S. Mincer1 and Steven D. Schwartz1,2 1Department of Biophysics, Albert Einstein College of Medicine, Bronx, NY, USA; 2Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA This review describes studies of particular enzymatically catalyzedreactionstoinvestigatethepossibilitythatcatalysis is mediated by protein dynamics. That is, evolution has crafted the protein backbone of the enzyme to direct vibra-tionsinsuchafashiontospeedreaction.Thereviewpresents the theoretical approach we have used to investigate this problem, but it is designed for the nonspecialist. The results show that in alcohol dehydrogenase, dynamic protein INTRODUCTION The transmission of an atom or group of atoms from the reactantregionofareactiontotheproductregionunderthe controlofanenzymeiscentraltobiochemistry.Themanner inwhichthe enzymespeedsthis transfer isin somecases still not clear. What is known is the end effect; enzymatic reactions occur at rates many orders of magnitude more rapid than the correspondingsolution phase reactions. This review will describe work recently completed in our group that has focused on examiningthe possibility that protein dynamicsmayinsomeenzymesplayacentralroleinhelping to produce the catalytic effect. These types of motions, which we have termed Ôrate promotingvibrations Õ, are motions of the protein matrix that change the geometry of the chemical barrier to reaction. By this we mean that both theheightandwidthofthebarrierarechanged.Thisunique role for the protein matrix has significant implications for the dynamics ofthechemical reaction; in particular, causing a barrier to narrow can significantly enhance a light particle’s ability to tunnel, while maskingthe normal kinetic indicators of such a phenomenon. It is this feature that we haveproposedasaunifyingprincipleforsomeexperimental data relatingto tunnelingin enzymatic reactions. This paper will describe our studies of rate promoting vibrations in enzymatic reactions with particular attention to the physical origins of the phenomenon. The structure of Correspondence to S. D. Schwartz, 1300 Morris Park Ave., Bronx, NY 10461, USA. Tel.: + 1 718 430 2139, E-mail: sschwartz@aecom.yu.edu Abbreviations:NAC,nearattackconformations;HLADH,horseliver alcohol dehydrogenase; YADH, yeast alcohol dehydrogenase. Note: a website is available at http://www.aecom.yu.edu/home/sggd/ faculty/schwartz.htm *Present address: Department of Chemical Engineering, Princeton University, NJ, USA. (Received 8 March 2002, revised 31 May 2002, accepted 6 June 2002) motion is in fact strongly coupled to chemical reaction in such a way as to promote catalysis. This result is in concert withbothexperimentaldataandinterpretationsforthisand other enzyme systems studied in the laboratories of the two other investigators who have published reviews in this issue. Keywords: protein dynamics; enzyme catalysis; tunneling; promotingvibration; promotingmode. this paper will be as follows: in the next section, we will briefly review a number of different potential mechanisms for enzyme catalytic action alongwith promotingvibra-tions. Followingthis, we will describe the mathematical foundation for our theories in some detail. This section will be written for nonexperts, but will contain the necessary formulae for the specialist as well. It will include the relationship between the current theories and a well-known approachtochargedparticletransferinbiologicalreactions, namely the Marcus theory. In this section we will also describe a simple nonbiological chemical system in which the physical features of promotingvibrations may be easily understood – proton transfer in organic acid crystals. We will then describe how we have used these concepts to fit seemingly anomalous kinetic data for enzymatic reactions. In the next section, we explore how one might rigorously identify the presence of such a promotingvibration in any enzymatic reaction, and illustrate the concepts with appli-cations to specific enzyme systems. The paper then con-cludes with discussions of future directions for this research. POTENTIAL MODES OF ENZYMATIC ACTION The exact physical mechanisms by which enzymes cause catalysis is still a topic for vigorous dialogue [1–3]. The research described in this paper will argue for a strong contribution from a nontraditional source, i.e. directed protein motions. In order to place this concept into a context, we will briefly review other potential mechanisms for enzymes to cause catalysis. We emphasize that none of these mechanisms are mutually exclusive, and are probably all involved in catalysis to a greater or lesser extent in each enzyme system. One of the earliest and still widely accepted ideas used to explainthiscatalyticefficiencyis thetransitionstate-binding concept of Pauling[4]. In this picture, as a chemical substance is beingtransformed from reactants to products, thespeciesthatbindsmoststronglytotheenzymeisatsome 3104 D. Antoniou et al. (Eur. J. Biochem. 269) intermediate point thought to be at or near the top of the solution phase (i.e. uncatalyzed) barrier to reaction. This preferential bindingreleases eneryg that stabilizes the transition state and thus lowers the barrier to reaction. This is a standard picture for nonbiological catalysis, and it also has significant experimental support. A critical observation is found usingkinetic isotope effect methods. In this way, one can probe the chemical structure of the transition state in the catalytic event. Stable molecules can be designed that sharetheelectronicpropertiesofthetransitionstate(usually identified by the electrostatic potential at the van der Waals surface). Furthermore, these molecules make highly potent inhibitors [5,6]. When substrate-like molecules that cannot react to form products bind, often a far lower level of inhibition is found. This result is said to be indicative of the fact that the transition state is strongly bound. It has been argued, however, that the electrostatic character of the active site duringthe catalytic event is largely determined by whatever charge stabilization is needed as the reaction progresses. If an inhibitor is designed with the complement-ary charges, it will bind strongly to the active site. However, this does not imply that the method by which the enzyme produced catalysis was transition state bindingand con-comitant release of energy [1]. A second approach, which might be viewed as the converse of transition state stabilization, is ground state destabilization.Inthispicture[7],theroleoftheenzymeisto make the reactants less stable rather than makingthe transitionstatemorestable.Thustheenergetichillthatmust be climbed with thermal activation is lowered. Energies are all relative and so the end effect of this and the first mechanism are the same; loweringthe relative eneryg difference between reactants and transition state. But it is clear that this view presents a very different physical mechanism. Recent calculations [8] seem to show that this model may well be dominant for the most efficient enzyme known, orotidine monophosphate decarboxylase. A third concept that has been also suggested. In solution, reactants are strongly solvated by water, the dominant component of most livingcells. When enzymes bind reactants, they often exclude water, and this lowered dielectric environment may be more conducive to reaction [9–11]. This approach to catalysis tends to treat the catalytic event much like an electron transfer reaction in solution. The dominant description of electron transfer in solution is Marcus’theory[12],andthisapproachhasalsobeenusedto describe atom transfer [13]. The concept here is that the main barrier to reaction is, in fact, reorganization of the solvent as charged particles move, rather than the intrinsic chemical barrier due to transformation of the substrate. It is certainly true that such energy reorganization may be a significantcomponentinmany cases,butprobablydoes not account for all catalysis in biological systems. A fourth recent suggestion by Bruice [14,15] is that the dominant role of an enzyme is to position substrates in such a way that thermal fluctuations easily take them over the barrier to reaction. The set of positions the enzyme encourages the substrate to take are known as Ônear attack conformationsÕ (NACs). Here, while the enzyme might bind strongly to a transition state structure,this binding energy is not thought to be released specifically to speed the reaction. The enzyme moulds the substrate so that it is on the edge of reactingand formingproducts. Because the enzyme helps Ó FEBS 2002 thereactantstoformtheNAC,thisviewisphilosophicallya bit closer to the ground state destabilization view. It is, however, not a statistical energetic argument, but rather a chemical structure argument. Afifthpossibilityforthemodeofactionofenzymesisthe principle subject of this paper, that is, motions within the protein itself actually speed the rate of a chemical reaction. There is significant relation between this possibility and the last view of catalysis described above, i.e. the creation of the NAC.Itmustbestressed,however,thatthecurrentviewisa dynamic one. For this concept to be true, actual motions of the protein must couple strongly to a reaction coordinate and cause an increase in reaction rate. This is not simply preparation of a reactive species, but rather dynamic coupling. It is important to note that this is an entirely different view of the method by which the enzyme accomplishes rate acceleration. In this view, evolution has created a protein structure that moves in such a way as to lowerabarrierandmakeitlesswide.Itmustbeemphasized that this loweringof the barrier is not statistical loweringof a potential of mean force through the release of binding energy, but rather the use of highly directed energy (a vibration) in a specific direction. Furthermore, this is not simply the statistical preparation ofreactive species as in the NAC concept. Here, protein dynamics directly affect the reaction coordinate potential. Although this effect can be quite apparent for a tunnelingsystem (the probability to tunnel increases exponentially with a reduction of the width of the tunnelingbarrier), it is equally important for systems where the reaction proceeds through classical transfer, because as the barrier is made narrower, it is also lowered. In order to understand how directed protein motions may cause catalysis, we need a theory of chemical reactions in a condensed phase. Our group has developed theories over the past 10 years, and this work, initially developed for simple condensed phases, such as polar media, forms the basis for our analysis. We now describe these theories in some detail. AN ENZYME AS A CONDENSED PHASE: THEORETICAL FORMULATION FOR THE STUDY OF CHEMICAL REACTION There are two requirements to enable the study of a chemical reaction in any system, be it as simple as a gas phase collision, or as complex as that in an enzyme. First, a potential energy for the interaction of all the atoms in the system is needed. This includes the interactions of all atoms havingtheirchemicalbondschanged,andthosethatdonot. The second requirement is for a method to solve the dynamics of the equations of motion that allow one to follow the progress of the reacting species in the presence of the rest of the system from reactants to products. In this work, we assume that we are able to obtain the first requirement (the potential). In order to study the dynamics on this potential, however, one needs to solve the Schro-dinger equation for the entire collection of atoms. It is a well-known fact that this is difficult for three or four atoms, andsoessentiallyimpossibleforthethousandsofatomsina reaction catalyzed by an enzyme. Various groups have taken a number of possible approaches to solve this problem. One may assume that quantum effects are minor, and use a purely classical Ó FEBS 2002 Barrier passage and protein dynamics (Eur. J. Biochem. 269) 3105 approach to solve the dynamics [16]. We are specifically interested in studies of enzyme systems where quantum mechanics plays a significant role, through tunneling of a light particle, in the chemical step of the enzyme, and so the classical approach will not be expected to yield valid results. Another approach is to use a mixed quantum-classical formulation in which a subset of the atoms is treated quantum mechanically and the rest of the system is treated purely classically. In recent years, this approach has become popular with the pioneeringwork of such investigators as Gao [8]. We have chosen a different approach, largely on stylistic grounds. Rather than treating the full collection of atoms as a mixture of quantum and classical objects (somethingthat is difficult to define rigorously), we have developed approximate approaches to treat the entire collection of atoms as a quantum mechanical entity. As mentionedabove,bothapproachesareapproximate,butwe prefer to make the approximation uniform for the entire system. We have called our approach the ÔQuantum KramersÕ methodology [17,18]. Our ideas were motivated by the followingapproximations developed for the study of the classical mechanics of large, complex systems. It is known that for a purely classical system [19,20], an accurate approximation of the dynamics of a tagged degree of freedom (for example a reaction coordinate) in a condensed phase can be obtained through the use of a generalized Langevin equation. The generalized Langevin equation is given by Newtonian dynamics plus the effects of the environmentintheformofamemoryfrictionandarandom force[21].Thus,allthecomplexmicroscopicdynamicsofall degrees of freedom other than the reaction coordinate are included only in a statistical treatment, and the reaction coordinate plus environment is treated as a modified one-dimensional system. What allows a realistic simulation of complex systemsis thatthestatistics ofthe environmentcan in fact be calculated from a formal prescription. This prescription is given by the Ôfluctuation-dissipation the-oremÕ, which yields the relationship between friction and random force. In particular, this theory enables us to calculate the memory friction from a relatively short-time classical simulation of the reaction coordinate. The Quan-tum Kramers approach, in turn, is dependent on an observation of Zwanzig[22,23]; if an interaction potential for a condensed phase system satisfies a fairly broad set of mathematical criteria, the dynamics of the reaction coordi-nate as described by the generalized Langevin equation can be rigorously equated to a microscopic Hamiltonian in which the reaction coordinate is coupled to an infinite set of Harmonic Oscillators via simple bilinear coupling: H ¼ P2 þVo þX P2 þ1mkx2q cks 2 ð1Þ s k k k k The first two terms in this Hamiltonian represent the kinetic and potential energy of the reaction coordinate, and the last set of terms similarly represent the kinetic and potential energy for an environmental bath. Here, s represents some coordinate that measures progress of the reaction (for example, in alcohol dehydrogenase where the chemical step is transfer of a hydride, s might be chosen to represent the relative position of the hydride from the alcohol to the NAD cofactor.) ck is the strength of the coupling of the environmental mode to the reaction coordinate, and mk and xk give the effective mass and frequency, respectively, of the environmental bath mode. A discrete spectral density gives the distribution of bath modes in the harmonic environment: JðxÞ ¼ p k mcxk ½dðx xkÞ dðx þ xkÞ ð2Þ Here d(x ) xk) is the ÔDirac deltaÕ function, so the spectral density is simply a collection of spikes, located at the frequency positions of the environmental modes, weighted by the strength of the coupling of these modes to the reaction coordinate. Note that this infinite collection of oscillators is purely fictitious; they are chosen to reproduce the overall physical properties of the system, but do not necessarily represent specific physical motions of the atoms in the system. It would seem that we have not made a huge amount of progress; we began with a many-dimensional system (classical) and found out that it could be accurately approximated by a one-dimensional system in a frictional environment (the generalized Langevin equation.) We have now recreated a many-dimensional system (the Zwanzig Hamiltonian). The reason we have done this is twofold. First, there is no true quantum mechanical analogue of friction, and so there really is no way to use the generalized Langevin approach for a quantum system, such as we would like to do for an enzyme. Second, the new quantum Hamiltonian given in Eqn (1) is much simpler than the Hamiltonian for the full enzymatic system. Harmonic oscillators are a problem that can easily be solved by quantum mechanics. Thus, the prescription is, given a potential for the enzymatic reaction, we model the exact problem usingZwanzigHamiltonian, as in Eqn (1), with the distribution of harmonic modes given by the spectral density in Eqn (2), and found through a simple classical computation of the frictional force on the reaction coordi-nate. Then, usingmethods to compute quantum dynamics developed in our group [24–29], quantities such as rates or kineticisotopeeffectsmaybecomputed.Thus,thequantum Kramers method, developed in our group, consists of the followingingredients. Given a potential for the enzymatic reaction, we model the exact problem usingZwanzi’gs Hamiltonian, as in Eqn (1), with the distribution of harmonic modes given by the spectral density in Eqn (2). The spectral density is obtained through a Ômolecular dynamicsÕ simulation of the classical system. Then, using methods developed in our group to carry out the quantum dynamics, quantities such as rates or kinetic isotope effects may be computed. Thisapproachenablesustomodelavarietyofcondensed phase chemical reactions with essentially experimental accuracy [30]. There are deeper connections between this approach and another popular method of dynamics com-putation in complex systems. We have shown [30] that this collection of bilinearly-coupled oscillators is in fact a microscopic version of the popular Marcus theory for chargedparticletransfer[12,13].Thebilinearcouplingofthe bathofoscillatorsisthesimplestformofaclassofcouplings that may be termed antisymmetric because of the mathe-matical property of the functional form of the couplingon reflection about the origin. This property has deeper implications than the mathematical nature of the symmetry 3106 D. Antoniou et al. (Eur. J. Biochem. 269) properties. Antisymmetric couplings, when coupled to a double-well-like potential energy profile, are able to instan-taneously change the level of well depths, but do nothing to thepositionofwellminima.Thismodulationintheposition of minima is exactly what the environment is envisaged to do within the Marcus theory paradigm. As we have shown [30], the minima of the total potential in Eqn (1) will occur, for a two-dimensional version of this potential, when the q degree of freedom is exactly equal and opposite in sign to cs2, and the minimum of the potential energy profile along the reaction coordinate is unaffected by this coupling. Within Marcus’ theory, which is a deep tunnelingtheory, transfer of the charged particle occurs at the value of the bath coordinates that cause the total potential to become symmetrized. Thus, ifthe bare reaction coordinate potential is symmetric, then the total potential is symmetrized at the position of the Ôbath plus couplingÕ minimum. When this configuration isachieved, theparticletunnels; theactivation energyforthereactionislargelytheenergytobringthebath into this favorable tunnelingconfiguration. While Marcus’ theory and our microscopic quantum Kramerstheoryarehighlysuccessfulinmanycases,inother cases, it is not possible to reproduce experimental results usingsuch an approach. The reason for this is that the antisymmetric couplingcontained within the Zwanzig Hamiltonian does not physically represent all possible important motions in a complex reactingsystem. In fact, such a reality was pointed out some time ago in seminal work of the Hynes group [31]. In some of our earlier work on hydrogen transfer in enzymatic systems, we were able to showthatonecouldreasonablyfitexperimentalkineticdata in such enzymatic systems with phenomenological applica-tion of the Hynes theories [32]. We became interested in a microscopic study of such systems in the examination of nonbiological proton transfer reactions, i.e. organic acid crystals. The simplest example is a carboxylic acid dimer, shown in Fig . 1. Such systems had been studied for many years [33–37], and they presented what seemed to be a chemical physics conundrum. While quantum chemistry computations seemed to show that the intrinsic barrier to proton transfer in these systems was reasonably high, and low experimental activation energies seemed to indicate a significant involvement of quantum tunneling in the proton transfer mechanism, careful measurements of kinetic iso-tope effects showed kinetics indicative of classical transfer. In order to study such systems, a rigorous theory, which allowed inclusion of symmetrically coupled vibrations, in addition to an environmental bath of antisymmetrically coupled oscillators, was needed. Mathematically, the simp-lest transformation of the Hamiltonian in Eqn (1) is given by: Ó FEBS 2002 H ¼ P2 þ Vo þ X P2 þ 1mkx2q cks 2 s k k þ 2M þ 1mX2Q Cs22 ð3Þ Note that in this case, the oscillator that is symmetrically coupled, represented by the last term in Eqn (3), is in fact a physical oscillation of the environment. We were able to develop a theory [38] of reactions mathematically represented by the Hamiltonian in Eqn (3), and usingthis method and experimentally available param-eters for the benzoic acid proton transfer potential, we were able to reproduce experimental kinetics as longas we included a symmetrically coupled vibration [39]. The results are shown in Table 1 below. The two-dimensional activa-tion energies refer to a two-dimensional system comprised of the reaction coordinate and a symmetrically coupled vibration. The reaction coordinate is also coupled to an infinite environment as described above. In this case, the symmetric motion has a clear physical origin: the symmetric motion of the carbonyl and hydroxyl oxygen atoms toward each other. Kinetic isotope effects in this system are modest, even though the vast majority of the proton transfer occurs via quantum tunneling. The end result of this study is that symmetrically coupled vibrations can significantly enhance rates of light particle transfer, and also significantly mask kinetic isotope signatures of tunnel-ing. A physical origin for this masking of the kinetic isotope effect may be understood from a comparison of the two-dimensional problem comprised of a reaction coordinate coupledsymmetricallyandantisymmetricallytoavibration. As Fig. 2 shows, antisymmetric coupling causes the minima (the reactants and products) to lie on a line; the minimum energy path, which passes through the transition state. In contrast, symmetric couplingcauses the reactants and products to be moved from the reaction coordinate axis in such a way that a straight line connection of reactant and products would pass no where near the transition state. This, in turn, results in the gas phase physical chemistry phenomenon known as corner cutting[40–42]. Physically, the quantity to be minimized alongany path from reactant to products is the action. This is an integral of the energy, andsolooselyspeaking,itisaproductofdistanceanddepth under the barrier that must be minimized to find an approximation to the tunnelingpath. The action also includes the mass of the particle beingtransferred, and so in the symmetric couplingcase, a proton will actually follow a very different physical path from reactants to products in a reaction than a deuteron. (Not just in the trivial sense that one tunnels more than another). It is this followingof a different physical path, even when tunnelingdominates, Table 1. Activation energies for H and D transfer in benzoic acid crystals at T ¼ 300 K. Three values are shown: the activation energies calculatedusingaone-andtwo-dimensionalKramersproblemandthe experimental values. The values of energies are in kcalÆmol)1. Fig. 1. A benzoic acid dimer. The reaction coordinate in this case is the symmetric transfer of the hydroxyl protons to the carbonyl oxygen. The promotingvibration is the symmetric motion of the oxyegns toward each other. E1d E2d H 3.39 1.51 D 5.21 3.14 Experiment 1.44 kcalÆmol)1 3.01 kcalÆmol)1 Ó FEBS 2002 Barrier passage and protein dynamics (Eur. J. Biochem. 269) 3107 q A s0 +s0 s A,S S Fig. 2. This diagram shows the location of stable minima in two-dimensional systems. In one case a vibrational mode is symmetrically coupled to the reaction coordinate, and in the other, antisymmetrically coupled. The figure represents how antisymmetrically and symmetri-cally coupled vibrations effect position of stable minima – that is reactantandproductwells–inmodulatingtheonedimensionaldouble well potential (before couplingalongthe x axis). The x axis, s, repre-sents the reaction coordinate, and q the coupled vibration. The points on the figure labeled S and A are the positions of the well minimal in the two dimensional system with symmetric and antisymmetric coup-ling, respectively. An antisymmetrically coupled vibration displaces those minima alonga straihgt line, so that the shortest distance between the reactant and product wells passes through the transition state. In contradistinction, a symmetrically coupled vibration, allows for the possibility of Ôcorner cuttingÕ under the barrier. For example, a proton and a deuteron will follow different paths under the barrier. that causes the kinetic isotope effects to be masked. It was thislowlevelofprimarykineticisotopeeffectthatsuggested a similarity between the proton transfer mechanism in the organic acid crystal and that of enzymatic reactions. While coupled motions of nearby atoms in enzymatic reactions have been used to explain anomalous kinetic isotope effects [43], these were studies in a classical picture with semiclas-sical tunnelingadded (the Bell correction; [44]) and they could not be used to account for enzymatic reactions in a deep tunnelingregime. Klinman and coworkers have helped pioneer the study of tunnelingin enzymatic reactions. One focus of their work has been the alcohol dehydrogenase family of enzymes. Alcohol dehydrogenases are NAD+-dependent enzymes that oxidize a wide variety of alcohols to the corresponding aldehydes. After successive bindingof the alcohol and cofactor, the first step is generally accepted to be complex-ation of the alcohol to one of the two bound Zinc ions [45]. ThiscomplexationlowersthepKa ofthealcoholprotonand causes the formation of the alcoholate. The chemical step is then transfer of a hydride from the alkoxide to the NAD+ cofactor. They [46] have found a remarkable effect on the kinetics of yeast alcohol dehydrogenase (a mesophile) and a related enzyme from Bacillus stereothermophilus, a thermo-phile. A variety of kinetic studies from this group have found that the mesophile [47] and many related dehydro-genases [48–51] show signs of significant contributions of quantum tunnelingin the rate-determiningstep of hydride transfer. Remarkably, their kinetic data seem to show that the thermophilic enzyme actually exhibits less signs of tunnelingat lower temperatures. Recent data of Kohen & Klinman [52] also show, via isotope exchange experiments, that the thermophile is significantly less flexible at mesophi-lic temperatures, as in the results of Petsko et al. [53], who conducted studies of 3-isopropylmalate dehydrogenase fromthethermophilicbacteriaThermusthermophilus.These data have been interpreted in terms of models similar to those we have described above, in which a specific type of proteinmotionstrongly promotes quantum tunneling; thus, at lower temperatures, when the thermophile has this motion significantly reduced, the tunneling component of reaction is hypothesizedtogodown eventhoughone would normally expect tunneling to go up as temperature goes down.Additionally,theKlinmangrouphasinvestigatedthe catalyticpropertiesofvariousmutantsofhorseliveralcohol dehydrogenase (HLADH). HLADH in the wild-type has a slightly less advantageous system to study than yeast alcohol dehydrogenase, because the chemistry is not the rate determiningstep in catalysis for this enzyme. Two specific mutations have been identified, Val203 fi Ala and Phe93 fi Trp, which significantly affect enzyme kinetics. Both residues are located at the active site; the valine impingesdirectlyonthefaceoftheNAD+ cofactordistalto the substrate alcohol. Modification of this residue to the smaller alanine significantly lowers both the catalytic efficiency of the enzyme, as compared to the wild-type, and also significantly lowers indicators of hydrogen tunnel-ing[54]. Phe93 is a residue in the alcohol bindingpocket. Replacementwith the larger tryptophanmakesitharder for the substrate to bind, but does not lower the indicators of tunneling[55]. Bruice’s recent molecular dynamics calcula-tions [56] produce results consistent with the concept that mutation of the valine changes protein dynamics, and it is this alteration,missinginthemutationatposition93, which in turn changes tunneling dynamics. (We note the recent experimental results from Klinman’s group [57] in which no decrease in tunnelingis seen as the temperature is raised.) A final set of enzymes now thought to exhibit dynamic protein control of tunnelinghydrogen transfer is that in the amine dehydrogenase family. Scrutton and coworkers have extensively studied these enzymes [58]. Though similarly named and havinga similar end effect as the alcohol dehydrogenases, they employ radically different chemistry. These enzymes catalyze the oxidative deamination of primary amines to aldehydes and free ammonia. In this case, however, rather than a chemical step of hydride transfer, the rate determiningchemical step is proton transfer; and in fact these enzymes catalyze a coupled electron proton transfer reaction. Electrons are coupled to some cofactor, for example, in the case of aromatic amine dehydrogenase, the cofactor is tryptophan-tryptophyl qui-none. Kinetic studies have shown that methylamine dehy-drogenase exhibits not only relatively large primary kinetic isotope effects (unlike the alcohol dehydrogenases), but also very strongtemperature dependence in the measured activation energy. This experimental data has been inter-preted as showingthat the enzyme works via a promoting vibration [59], as we have suggested for bovine serum amine oxidase [32], and for various forms of HLADH [60]. Here, the primary kinetic isotope effect is 17, rather than 3 or 4. ... - tailieumienphi.vn