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Amnon Kohen - One of the best experts on this subject based on the ideXlab platform.
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role of dynamics in Enzyme Catalysis substantial versus semantic controversies
Accounts of Chemical Research, 2015Co-Authors: Amnon KohenAbstract:ConspectusThe 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 dynamic...
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linking protein motion to Enzyme Catalysis
Molecules, 2015Co-Authors: Priyanka Singh, Thelma Abeysinghe, Amnon KohenAbstract:Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, Catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of Enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of Enzymes—dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)—and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.
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hydrogen tunneling links protein dynamics to Enzyme Catalysis
Annual Review of Biochemistry, 2013Co-Authors: Judith P. Klinman, Amnon KohenAbstract: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 subs...
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hydrogen tunneling links protein dynamics to Enzyme Catalysis
Annual Review of Biochemistry, 2013Co-Authors: Judith P. Klinman, Amnon KohenAbstract: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 subs...
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Enzyme Catalysis beyond classical paradigms
ChemInform, 1998Co-Authors: Amnon Kohen, Judith P. KlinmanAbstract:Despite many decades of intense study, a full description of Enzyme Catalysis at the molecular level remains to be achieved. A number of aspects of bioCatalysis are widely accepted, including (i) the conversion of a chemical reaction from an inter- to an intramolecular process with the concomitant decrease in the entropy of activation and (ii) the stabilization of the transition state (TS) by the precise orientation of multiple functional groups at the Enzyme active site. These functional groups perform the roles of general acid/base, electrophilic/nucleophilic Catalysis and charge neutralization via electrostatic and H-bonding interactions. The process of covalent bond breaking and forming in Enzyme Catalysis is accompanied by substrate binding, product release, and protein rearrangement steps, which are rate determining for many Enzymes. The resulting, multibarrier
Judith P. Klinman - One of the best experts on this subject based on the ideXlab platform.
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dynamically achieved active site precision in Enzyme Catalysis
Accounts of Chemical Research, 2015Co-Authors: Judith P. KlinmanAbstract:ConspectusThe grand challenge in enzymology is to define and understand all of the parameters that contribute to Enzymes’ enormous rate accelerations. The property of hydrogen tunneling in Enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in Catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize Enzyme–substrate interactions and beyond.This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either e...
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hydrogen tunneling links protein dynamics to Enzyme Catalysis
Annual Review of Biochemistry, 2013Co-Authors: Judith P. Klinman, Amnon KohenAbstract: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 subs...
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hydrogen tunneling links protein dynamics to Enzyme Catalysis
Annual Review of Biochemistry, 2013Co-Authors: Judith P. Klinman, Amnon KohenAbstract: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 subs...
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Enzyme Catalysis beyond classical paradigms
ChemInform, 1998Co-Authors: Amnon Kohen, Judith P. KlinmanAbstract:Despite many decades of intense study, a full description of Enzyme Catalysis at the molecular level remains to be achieved. A number of aspects of bioCatalysis are widely accepted, including (i) the conversion of a chemical reaction from an inter- to an intramolecular process with the concomitant decrease in the entropy of activation and (ii) the stabilization of the transition state (TS) by the precise orientation of multiple functional groups at the Enzyme active site. These functional groups perform the roles of general acid/base, electrophilic/nucleophilic Catalysis and charge neutralization via electrostatic and H-bonding interactions. The process of covalent bond breaking and forming in Enzyme Catalysis is accompanied by substrate binding, product release, and protein rearrangement steps, which are rate determining for many Enzymes. The resulting, multibarrier
Stephen J Benkovic - One of the best experts on this subject based on the ideXlab platform.
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perspectives on electrostatics and conformational motions in Enzyme Catalysis
Accounts of Chemical Research, 2015Co-Authors: Philip Hanoian, Sharon Hammesschiffer, Tony C Liu, Stephen J BenkovicAbstract:CONSPECTUS: Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of Enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on Enzyme Catalysis in the Enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other Enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel Enzymes as well as those seeking to develop therapeutic agents that function as Enzyme inhibitors.
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flexibility diversity and cooperativity pillars of Enzyme Catalysis
Biochemistry, 2011Co-Authors: Gordon G Hammes, Stephen J Benkovic, Sharon HammesschifferAbstract:This brief review discusses our current understanding of the molecular basis of Enzyme Catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, nuclear magnetic resonance, and single-molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all Enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the Enzyme–substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for Enzyme Catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conforma...
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a dynamic knockout reveals that conformational fluctuations influence the chemical step of Enzyme Catalysis
Science, 2011Co-Authors: Gira Bhabha, Stephen J Benkovic, Jeeyeon Lee, D C Ekiert, Jongsik Gam, Ian A Wilson, Jane H Dyson, Peter E WrightAbstract:Conformational dynamics play a key role in Enzyme Catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of Enzyme Catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the Enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of Enzyme mechanisms and for design of novel protein catalysts.
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coupled motions in Enzyme Catalysis
Current Opinion in Chemical Biology, 2010Co-Authors: Vishal C Nashine, Sharon Hammesschiffer, Stephen J BenkovicAbstract:Recently, the hypothesis that protein motions are involved in enzymatic turnover has gained significant attention. We review cases where there is evidence that protein motions are rate-limiting in the overall catalytic cycle and examine experimental and theoretical evidence for how such motions enhance the probability of sampling the transition state configurations relative to the ground state. The impact of tunneling, the possible role of vibrational coupling and the value of conformational chemical landscapes are also scrutinized.
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free energy landscape of Enzyme Catalysis
Biochemistry, 2008Co-Authors: Stephen J Benkovic, Gordon G Hammes, Sharon HammesschifferAbstract:The concept is developed that Enzyme mechanisms should be viewed as “catalytic networks” with multiple conformations that occur serially and in parallel in the mechanism. These coupled ensembles of conformations require a multi-dimensional standard free-energy surface that is very “rugged”, containing multiple minima and transition states. Experimental and theoretical evidence is presented to support this concept.
Frances H Arnold - One of the best experts on this subject based on the ideXlab platform.
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dual function Enzyme Catalysis for enantioselective carbon nitrogen bond formation
Nature Chemistry, 2021Co-Authors: Zhen Liu, Carla Calvotusell, Andrew Z Zhou, Kai Chen, Marc Garciaborras, Frances H ArnoldAbstract:Chiral amines can be made by insertion of a carbene into an N–H bond using two-catalyst systems that combine a transition metal-based carbene-transfer catalyst and a chiral proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N–H insertion, but asymmetric protonation in an active site replete with proton sources is challenging. Here we describe engineered cytochrome P450 Enzymes that catalyse carbene N–H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to 32,100 total turnovers, >99% yield and 98% e.e.). These Enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this transformation and provide stereocontrol. A haem protein that serves as a dual-function catalyst capable of inserting a carbene into a N–H bond to form α-amino lactones has been reported. The Enzyme catalyses both carbene transfer and the subsequent proton transfer in a single active site. This transformation can proceed at the gram scale with high efficiency and enantioselective control.
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dual function Enzyme Catalysis for enantioselective carbon nitrogen bond formation
ChemRxiv, 2021Co-Authors: Zhen Liu, Carla Calvotusell, Andrew Z Zhou, Kai Chen, Marc Garciaborras, Frances H ArnoldAbstract:Whereas enzymatic asymmetric carbene N–H insertion is a powerful method for preparation of chiral amines in principle, it has suffered from limited enantioselectivity in practice. In this work, we demonstrate that engineered cytochrome P450 Enzymes can catalyze this abiological C–N bond-forming reaction with excellent activity and selectivity (up to 32,100 TTN, >99% yield and 98% e.e.) to prepare a series of bioactive α-amino lactones, which have not been accessed previously using a carbene insertion strategy. The Enzymes are dual-function catalysts, effecting both carbene transfer and enantioselective proton-transfer Catalysis, in a single active site. To gain insight into the mechanism of the enzymatic transformation, especially in the asymmetric protonation step, we performed extensive molecular dynamics simulations and density functional theory (DFT) calculations. Computational studies uncover the important roles of active-site residues that enable high activity and selectivity through interacting with the carbene intermediate and the amine substrate, and directing water molecules for selective proton transfer.
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anti markovnikov alkene oxidation by metal oxo mediated Enzyme Catalysis
Science, 2017Co-Authors: Stephan C Hammer, Grzegorz Kubik, Ella Watkins, Shan Huang, Hannah Minges, Frances H ArnoldAbstract:Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 Enzyme by directed evolution to catalyze metal-oxo–mediated anti-Markovnikov oxidation of styrenes with high efficiency. The Enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.
Adrian J Mulholland - One of the best experts on this subject based on the ideXlab platform.
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teaching Enzyme Catalysis using interactive molecular dynamics in virtual reality
Journal of Chemical Education, 2019Co-Authors: Simon J Bennie, Kara E Ranaghan, Adrian J Mulholland, Helen M Deeks, Heather E Goldsmith, Michael B Oconnor, David R GlowackiAbstract:The reemergence of virtual reality (VR) in the past few years has led to affordable, high-quality commodity hardware that can offer new ways to teach, communicate, and engage with complex concepts....
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differential transition state stabilization in Enzyme Catalysis quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field
Journal of the American Chemical Society, 2004Co-Authors: Borys Szefczyk, Kara E Ranaghan, Adrian J Mulholland, Andrzej W SokalskiAbstract:Chorismate mutase is a key model system in the development of theories of Enzyme Catalysis. To analyze the physical nature of catalytic interactions within the Enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the Enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that Catalysis by this Enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the Enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the Enzyme has evolved specifically to stabilize the TS relative to the substrate.
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transition state stabilization and substrate strain in Enzyme Catalysis ab initio qm mm modelling of the chorismate mutase reaction
Organic and Biomolecular Chemistry, 2004Co-Authors: Kara E Ranaghan, Lars Ridder, Borys Szefczyk, Andrzej W Sokalski, Johannes C Hermann, Adrian J MulhollandAbstract:To investigate fundamental features of Enzyme Catalysis, there is a need for high-level calculations capable of modelling crucial, unstable species such as transition states as they are formed within Enzymes. We have modelled an important model Enzyme reaction, the Claisen rearrangement of chorismate to prephenate in chorismate mutase, by combined ab initio quantum mechanics/molecular mechanics (QM/MM) methods. The best estimates of the potential energy barrier in the Enzyme are 7.4–11.0 kcal mol−1 (MP2/6-31+G(d)//6-31G(d)/CHARMM22) and 12.7–16.1 kcal mol−1 (B3LYP/6-311+G(2d,p)//6-31G(d)/CHARMM22), comparable to the experimental estimate of ΔH‡ = 12.7 ± 0.4 kcal mol−1. The results provide unequivocal evidence of transition state (TS) stabilization by the Enzyme, with contributions from residues Arg90, Arg7, and Arg63. Glu78 stabilizes the prephenate product (relative to substrate), and can also stabilize the TS. Examination of the same pathway in solution (with a variety of continuum models), at the same ab initio levels, allows comparison of the catalyzed and uncatalyzed reactions. Calculated barriers in solution are 28.0 kcal mol−1 (MP2/6-31+G(d)/PCM) and 24.6 kcal mol−1 (B3LYP/6-311+G(2d,p)/PCM), comparable to the experimental finding of ΔG‡ = 25.4 kcal mol−1 and consistent with the experimentally-deduced 106-fold rate acceleration by the Enzyme. The substrate is found to be significantly distorted in the Enzyme, adopting a structure closer to the transition state, although the degree of compression is less than predicted by lower-level calculations. This apparent substrate strain, or compression, is potentially also catalytically relevant. Solution calculations, however, suggest that the catalytic contribution of this compression may be relatively small. Consideration of the same reaction pathway in solution and in the Enzyme, involving reaction from a ‘near-attack conformer’ of the substrate, indicates that adoption of this conformation is not in itself a major contribution to Catalysis. Transition state stabilization (by electrostatic interactions, including hydrogen bonds) is found to be central to Catalysis by the Enzyme. Several hydrogen bonds are observed to shorten at the TS. The active site is clearly complementary to the transition state for the reaction, stabilizing it more than the substrate, so reducing the barrier to reaction.
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insights into Enzyme Catalysis from qm mm modelling transition state stabilization in chorismate mutase
Molecular Physics, 2003Co-Authors: Kara E Ranaghan, Lars Ridder, Borys Szefczyk, Andrzej W Sokalski, Johannes C Hermann, Adrian J MulhollandAbstract:Chorismate mutase provides an important test of theories of Enzyme Catalysis, and of modelling methods. The Claisen rearrangement of chorismate to prephenate in the Enzyme has been modelled here by a combined quantum mechanics/molecular mechanics (QM/MM) method. Several pathways have been calculated. The sensitivity of the results to details of model preparation and pathway calculation is tested, and the results are compared in detail to previous similar studies and experiments. The potential energy barrier for the Enzyme reaction is estimated at 24.5—31.6 kcal mol−1 (AMl/CHARMM), and 2.7—11.9 kcal mol−1 with corrections (e.g. B3LYP/6-31 + G(d)). In agreement with previous studies, the present analysis of the calculated paths provides unequivocal evidence of significant transition state stabilization by the Enzyme, indicating that this is central to Catalysis by the Enzyme. The active site is exquisitely complementary to the transition state, stabilizing it more than the substrate, so reducing the barrier...