Amnon Kohen

Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 °C; this is analogous to previous findings with mesophilic ADH at 25 °C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

Isotope Effects In Chemistry and Biology

Editorial Biography Theoretical Basis of Isotope Effects from an Autobiographical Perspective J. Bigeleisen Enrichment of Isotopes T. Ishida and Y. Fujii Comments on Selected Topics in Isotope Theoretical Chemistry M. Wolfsberg Condensed Matter Isotope Effects W.A. Van Hook Anharmonicities, Isotopes, and IR and NMR Properties of Hydrogen-Bonded Complexes J.E. Del Bene Isotope Effects on Hydrogen-Bond Symmetrization in Ice and Strong Acids at High Pressure K. Aoki Hydrogen Bond Isotope Effects Studied by NMR H-H. Limbach, G.S. Denisov and N.S. Golubev Isotope Effects and Symmetry of Hydrogen Bonds in Solution: Single- and Double-Well Potential J.S. Lau and C.L. Perrin NMR Studies of Isotope Effects of Compounds with Intramolecular Hydrogen Bonds P.E. Hansen Vibrational Isotope Effects in Hydrogen Bonds Z. Mielke and L. Sobczyk Isotope Selective Infrared Spectroscopy and Intramolecular Dynamics M. Hippler and M. Quack Nonmass-Dependent Isotope Effects R.E. Weston, Jr. Isotope Effects in the Atmosphere E. Roth, R. Letolle, C. M. Stevens, and F. Robert Isotope Effects for Exotic Nuclei O. Matsson Muonium - An Ultra-Light Isotope of Hydrogen E. Roduner The Kinetic Isotope Effect in the Photo-Dissociation Reaction of Excited-State Acids in Aqueous Solutions E. Pines The Role of an Internal-Return Mechanism on Measured Isotope Effects H.F. Koch Vibrationally Enhanced Tunneling and Kinetic Isotope Effects in Enzymatic Reactions S.D. Schwartz Kinetic Isotope Effects for Proton-Coupled Electron Transfer Reactions S. Hammes-Schiffer Kinetic Isotope Effects in Multiple Proton Transfer Z. Smedarchina, W. Siebrand, and A. Fernandez-Ramos Interpretation of Primary Kinetic Isotope Effects for Adiabatic and Nonadiabatic Proton-Transfer Reactions in a Polar Environment P.M. Kiefer and J.T. Hynes Variational Transition-State Theory and Multidimensional Tunneling for Simple and Complex Reactions in the Gas Phase, Solids, Liquids, and Enzymes D.G. Truhlar Computer Simulations of Isotope Effects in Enzyme Catalysis A. Warshel, Mats H. M. Olsson, and J. Villa-Freixa Chapter 24 Oxygen-18 Isotope Effects as a Probe of Enzymatic Activation of Molecular Oxygen Justine P. Roth and Judith P. Klinman Chapter 25 Solution and Computational Studies of Kinetic Isotope Effects in Flavoprotein and Quinoprotein Catalyzed Substrate Oxidations as Probes of Enzymic Hydrogen Tunneling and Mechanism J. Basran, L. Masgrau, M.J. Sutcliffe, and N.S. Scrutton Proton Transfer and Proton Conductivity in Condensed Matter Environment A.M. Kuznetsov and J. Ulstrup Mechanisms of CH-Bond Cleavage Catalyzed by Enzymes W. Siebrand and Z. Smedarchina Kinetic Isotope Effects as Probes for Hydrogen Tunneling in Enzyme Catalysis A. Kohen Hydrogen Bonds, Transition-State Stabilization, and Enzyme Catalysis R.L. Schowen Substrate and pH Dependence of Isotope Effects in Enzyme Catalyzed Reactions W.E. Karsten and P.F. Cook Catalysis by Alcohol Dehydrogenases B.V. Plapp Effects of High Hydrostatic Pressure on Isotope Effects D.B. Northrop Solvent Hydrogen Isotope Effects in Catalysis by Carbonic Anhydrase: Proton Transfer through Intervening Water Molecules D.N. Silverman and I. Elder Isotope Effects from Partitioning of Intermediates in Enzyme-Catalyzed Hydroxylation Reactions Paul F. Fitzpatrick Chlorine Kinetic Isotope Effects on Biological Systems P. Paneth Nucleophile Isotope Effects V.E. Anderson, A.G. Cassano, and M.E. Harris Enzyme Mechanisms from Isotope Effects W. W. Cleland Catalysis and Regulation in the Soluble Methane Monooxygenase System: Applications of Isotopes and Isotope Effects J.D. Lipscomb Secondary Isotope Effects A.C. Hengge Isotope Effects in the Characterization of Low Barrier Hydrogen Bonds P.A. Frey Theory and Practice of Solvent Isotope Effects D.M. Quinn Enzymatic Binding Isotope Effects and the Interaction of Glucose with Hexokinase B.E. Lewis and V.L. Schramm Index

Hydrogen tunneling in biology

The mechanistic details of hydrogen transfer in biological systems are not fully understood. The traditional approach has been to use semiclassical transition-state theory. This theory cannot explain many experimental findings, however, so different approaches that emphasize the importance of quantum mechanics and dynamic effects should also be considered.

Hydrogen tunneling links protein dynamics to enzyme catalysis.

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase.

One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes a single C-H–C transfer, is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor–acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry.

Arrhenius curves of hydrogen transfers: tunnel effects, isotope effects and effects of pre-equilibria

In this paper, the Arrhenius curves of selected hydrogen-transfer reactions for which kinetic data are available in a large temperature range are reviewed. The curves are discussed in terms of the one-dimensional Bell–Limbach tunnelling model. The main parameters of this model are the barrier heights of the isotopic reactions, barrier width of the H-reaction, tunnelling masses, pre-exponential factor and minimum energy for tunnelling to occur. The model allows one to compare different reactions in a simple way and prepare the kinetic data for more-dimensional treatments. The first type of reactions is concerned with reactions where the geometries of the reacting molecules are well established and the kinetic data of the isotopic reactions are available in a large temperature range. Here, it is possible to study the relation between kinetic isotope effects (KIEs) and chemical structure. Examples are the tautomerism of porphyrin, the porphyrin anion and related compounds exhibiting intramolecular hydrogen bonds of medium strength. We observe pre-exponential factors of the order of kT/h≅1013 s−1 corresponding to vanishing activation entropies in terms of transition state theory. This result is important for the second type of reactions discussed in this paper, referring mostly to liquid solutions. Here, the reacting molecular configurations may be involved in equilibria with non- or less-reactive forms. Several cases are discussed, where the less-reactive forms dominate at low or at high temperature, leading to unusual Arrhenius curves. These cases include examples from small molecule solution chemistry like the base-catalysed intramolecular H-transfer in diaryltriazene, 2-(2′-hydroxyphenyl)-benzoxazole, 2-hydroxy-phenoxyl radicals, as well as in the case of an enzymatic system, thermophilic alcohol dehydrogenase. In the latter case, temperature-dependent KIEs are interpreted in terms of a transition between two regimes with different temperature-independent KIEs.

Role of Dynamics in Enzyme Catalysis: Substantial versus Semantic Controversies

Conspectus The role of the enzyme’s dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term “protein dynamics” is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is “stored” in a specific protein mode and “used” for catalysis before it can dissipate to its environment (i.e., “nonstatistical dynamics”). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual “induced-fit” shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction’s transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions.

Effects of the Donor–Acceptor Distance and Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed Reaction

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor-acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DADs rearrangement toward tunneling-ready states. This structure-function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C-H→C transfer reactions.

An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene.

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2′-deoxyuridine-5′-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2′-deoxyuridine-5′-monophosphate. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (that is, a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2′-deoxythymidine-5′-monophosphate. These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

KINETIC ISOTOPE EFFECTS AS PROBES FOR HYDROGEN TUNNELING, COUPLED MOTION AND DYNAMICS CONTRIBUTIONS TO ENZYME CATALYSIS

Since the early days of enzymology attempts have been made to deconvolute the various contributions of physical phenomena to enzyme catalysis. Here we present experimental and theoretical studies that examine the possible role of hydrogen tunneling, coupled motion, and enzyme dynamics in catalysis. In this review, we first introduce basic concepts of enzyme catalysis from a physical chemistry point of view. Then, we present several recent developments in the application of experimental tools that can probe tunneling, coupled motion, dynamic effects and other possible physical phenomena that may contribute to catalysis. These tools include kinetic isotope effects (KIEs), their temperature dependency and H/D/T mutual relations (the Swain–Schaad relationship). Several theories and models that assist in understanding those phenomena are also described. The possibility that these models invoke a direct role for the enzymes dynamics (environmental fluctuations and rearrangements) is discussed. Finally, the need to compare the enzymatic reaction to the uncatalyzed one while investigating contributions to catalysis is emphasised.