Preorganization and reorganization as related factors in enzyme catalysis: the chorismate mutase case

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.     

Structural reorganization and preorganization in enzyme active sites: comparisons of experimental and theoretically ideal active site geometries in the multistep serine esterase reaction cycle

Many enzymes catalyze reactions with multiple chemical steps, requiring the stabilization of multiple transition states during catalysis. Such enzymes must strike a balance between the conformational reorganization required to stabilize multiple transition states of a reaction and the confines of a preorganized active site in the polypeptide tertiary structure. Here we investigate the compromise between structural reorganization during the catalytic process and preorganization of the active site for a multistep enzyme-catalyzed reaction, the hydrolysis of esters by the Ser-His-Asp/Glu catalytic triad. Quantum mechanical transition states were used to generate ensembles of geometries that can catalyze each individual step in the mechanism. These geometries are compared to each other by superpositions of catalytic atoms to find "consensus" geometries that can catalyze all steps with minimal rearrangement. These consensus geometries are found to be excellent matches for the natural active site. Preorganization is therefore found to be the major defining characteristic of the active site, and reorganizational motions often proposed to promote catalysis have been minimized. The variability of enzyme active sites observed by X-ray crystallography was also investigated empirically. A catalog of geometrical parameters relating active site residues to each other and to bound inhibitors was collected from a set of crystal structures. The crystal-structure-derived values were then compared to the ranges found in quantum mechanically optimized structures along the entire reaction coordinate. The empirical ranges are found to encompass the theoretical ranges when thermal fluctuations are taken into account. Therefore, the active sites are preorganized to a geometry that can be objectively and quantitatively defined as minimizing conformational reorganization while maintaining optimal transition state stabilization for every step during catalysis. The results provide a useful guiding principle for de novo design of enzymes with multistep mechanisms.     

Analysis of chorismate mutase catalysis by QM/MM modelling of enzyme-catalysed and uncatalysed reactions

Chorismate mutase is at the centre of current controversy about fundamental features of biological catalysts. Some recent studies have proposed that catalysis in this enzyme does not involve transition state (TS) stabilization but instead is due largely to the formation of a reactive conformation of the substrate. To understand the origins of catalysis, it is necessary to compare equivalent reactions in different environments. The pericyclic conversion of chorismate to prephenate catalysed by chorismate mutase also occurs (much more slowly) in aqueous solution. In this study we analyse the origins of catalysis by comparison of multiple quantum mechanics/molecular mechanics (QM/MM) reaction pathways at a reliable, well tested level of theory (B3LYP/6-31G(d)/CHARMM27) for the reaction (i) in Bacillus subtilis chorismate mutase (BsCM) and (ii) in aqueous solvent. The average calculated reaction (potential energy) barriers are 11.3 kcal mol(-1) in the enzyme and 17.4 kcal mol(-1) in water, both of which are in good agreement with experiment. Comparison of the two sets of reaction pathways shows that the reaction follows a slightly different reaction pathway in the enzyme than in it does in solution, because of a destabilization, or strain, of the substrate in the enzyme. The substrate strain energy within the enzyme remains constant throughout the reaction. There is no unique reactive conformation of the substrate common to both environments, and the transition state structures are also different in the enzyme and in water. Analysis of the barrier heights in each environment shows a clear correlation between TS stabilization and the barrier height. The average differential TS stabilization is 7.3 kcal mol(-1) in the enzyme. This is significantly higher than the small amount of TS stabilization in water (on average only 1.0 kcal mol(-1) relative to the substrate). The TS is stabilized mainly by electrostatic interactions with active site residues in the enzyme, with Arg90, Arg7 and Glu78 generally the most important. Conformational effects (e.g. strain of the substrate in the enzyme) do not contribute significantly to the lower barrier observed in the enzyme. The results show that catalysis is mainly due to better TS stabilization by the enzyme.     

Differential transition-state stabilization in enzyme catalysis: quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field

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.     

Crucial role of protein flexibility in formation of a stable reaction transition state in an α-amylase catalysis

Conformational flexibility of proteins provides enzymes with high catalytic activity. Although the conformational flexibility is known to be pivotal for the ligand binding and release, its role in the chemical reaction process of the reactive substrate remains unclear. We determined a transition state of an enzymatic reaction in a psychrophilic α-amylase by a hybrid molecular simulation that allows one to identify the optimal chemical state in an extensive conformational ensemble of protein. The molecular simulation uncovered that formation of the reaction transition state accompanies a large and slow movement of a loop adjacent to the catalytic site. Free energy calculations revealed that, although catalytic electrostatic potentials on the reactive moiety are formed by local and fast reorganization around the catalytic site, reorganization of the large and slow movement of the loop significantly contributes to reduction of the free energy barrier by stabilizing the local reorganization.     

Perspective: pre-chemistry conformational changes in DNA polymerase mechanisms

In recent papers, there has been a lively exchange concerning theories for enzyme catalysis, especially the role of protein dynamics/pre-chemistry conformational changes in the catalytic cycle of enzymes. Of particular interest is the notion that substrate-induced conformational changes that assemble the polymerase active site prior to chemistry are required for DNA synthesis and impact fidelity (i.e., substrate specificity). High-resolution crystal structures of DNA polymerase β representing intermediates of substrate complexes prior to the chemical step are available. These structures indicate that conformational adjustments in both the protein and substrates must occur to achieve the requisite geometry of the reactive participants for catalysis. We discuss computational and kinetic methods to examine possible conformational change pathways that lead from the observed crystal structure intermediates to the final structures poised for chemistry. The results, as well as kinetic data from site-directed mutagenesis studies, are consistent with models requiring pre-chemistry conformational adjustments in order to achieve high fidelity DNA synthesis. Thus, substrate-induced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual rate-determining steps for chemistry.     

Role of protein dynamics in reaction rate enhancement by enzymes

An integrated view of protein structure, dynamics, and function is emerging, where proteins are considered as dynamically active assemblies and internal motions are closely linked to function such as enzyme catalysis. Further, the motion of solvent bound to external regions of protein impacts internal motions and, therefore, protein function. Recently, we discovered a network of protein vibrations in enzyme cyclophilin A, coupled to its catalytic activity of peptidyl-prolyl cis-trans isomerization. Detailed studies suggest that this network, extending from surface regions to active site, is a conserved part of enzyme structure and has a role in promoting catalysis. In this report, theoretical investigations of concerted conformational fluctuations occurring on microsecond and longer time scales within the discovered network are presented. Using a new technique, kinetic energy was added to protein vibrational modes corresponding to conformational fluctuations in the network. The results reveal that protein dynamics promotes catalysis by altering transition state barrier crossing behavior of reaction trajectories. An increase in transmission coefficient and number of productive trajectories with increasing amounts of kinetic energy in vibrational modes is observed. Variations in active site enzyme-substrate interactions near transition state are found to be correlated with barrier recrossings. Simulations also showed that energy transferred from first solvation shell to surface residues impacts catalysis through network fluctuations. The detailed characterization of network presented here indicates that protein dynamics plays a role in rate enhancement by enzymes. Therefore, coupled networks in enzymes have wide implications in understanding allostericity and cooperative effects, as well as protein engineering and drug design.     

Probing single-molecule enzyme active-site conformational state intermittent coherence

The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.     

Transition state stabilization and substrate strain in enzyme catalysis: ab initio QM/MM modelling of the chorismate mutase reaction

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 Delta H(++)= 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 Delta G(++)= 25.4 kcal mol(-1) and consistent with the experimentally-deduced 10(6)-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.     

Ribonucleotide activation by enzyme ribonucleotide reductase: understanding the role of the enzyme

This article focuses on the first step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This corresponds to the activation of the substrate. In this work a large model of the active site region involving 130 atoms was used instead of the minimal gas phase models used in previous works. The ONIOM method was employed to deal with such a large system. The results gave additional information, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme in this step. Enzyme-substrate interaction energies, specific transition state stabilization, and substrate steric strain energies were obtained. It was concluded that the transition state is stabilized in 4.0 kcal/mol by specific enzyme-substrate interactions. However, this stabilization is cancelled by the cost in conformational energy for the enzyme to adopt the transition state geometry; the overall result is that the enzyme machinery does not lead to a rate enhancement in this step. It was also found that the substrate binds to the active site with almost no steric strain, emphasizing the complementarity and specificity of the RNR active site for nucleotide binding. The main role of the enzyme at the very beginning of the catalytic cycle was concluded to be to impose stereospecifity upon substrate activation and to protect the enzyme radical from the solvent, rather than to be an reaction rate enhancement.     

Microsecond timescale MD simulations at the transition state of PmHMGR predict remote allosteric residues

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to reach the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach to the second hydride transfer transition state of HMG-CoA reductase from Pseudomonas mevalonii (PmHMGR) identified three remote residues, R396, E399 and L407, (15–27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.

Transition state force fields enable MD simulations at the transition state of HMGCoA reductase that sample the transition state ensemble on the μs timescale to identify remote residues that affect the reaction rate.     

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate‐dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.     

Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5′‐phosphate (PLP)‐dependent D ‐ornithine 4,5‐aminomutase (OAM)‐catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff‐base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial “strain” energy on the orientation of the cyclic intermediate to control its trajectory. In addition the “strain” energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP‐dependent reactions.     

Allosteric Regulation of Enzymatic Reactions in a Transparent Inorganic Sol-Gel Material

Glutamate dehydrogenase is encapsulated in a transparent porous silicate matrix by using sol-gel techniques. The inorganic polymer is formed around the enzyme (MW > 300,000 D). The enzyme is active in the material, catalyzes the reaction of L-glutamate to 2-oxoglutarate and follows Michaelis-Menten kinetics. The allosteric regulators ADP and GTP inhibit or activate the reaction; at pH 6, GTP acts as a strong activator and ADP acts as an inhibitor. This system involves a complex series of interactions; the co-enzyme NAD⁺ is required for catalysis, large-scale conformational changes accompany the binding of the substrate and coenzyme to the enzyme, the activators/inhibitors must bind to the enzyme to regulate the reactions, and the substrates and products must diffuse through the matrix to and from the binding site. The influence of the unique matrix on the complex enzymatic system is discussed.     

A theoretical study of the catalytic mechanism of formate dehydrogenase

A theoretical study of the hydride transfer between formate anion and nicotinamide adenine dinucleotide (NAD(+)) catalyzed by the enzyme formate dehydrogenase (FDH) has been carried out by a combination of two hybrid quantum mechanics/molecular mechanics techniques: statistical simulation methods and internal energy minimizations. Free energy profiles, obtained for the reaction in the enzyme active site and in solution, allow obtaining a comparative analysis of the behavior of both condensed media. Moreover, calculations of the reaction in aqueous media can be used to probe the dramatic differences between reactants state in the enzyme active site and in solution. The results suggest that the enzyme compresses the substrate and the cofactor into a conformation close to the transition structure by means of favorable interactions with the amino acid residues of the active site, thus facilitating the relative orientation of donor and acceptor atoms to favor the hydride transfer. Moreover, a permanent field created by the protein reduces the work required to reach the transition state (TS) with a concomitant polarization of the cofactor that would favor the hydride transfer. In contrast, in water the TS is destabilized with respect to the reactant species because the polarity of the solute diminishes as the reaction proceeds, and consequently the reaction field, which is created as a response to the change in the solute polarity, is also decreased. Therefore protein structure is responsible of both effects; substrate preorganization and TS stabilization thus diminishing the activation barrier. Because of the electrostatic features of the catalyzed reaction, both media preferentially stabilize the ground-state, thus explaining the small rate constant enhancement of this enzyme, but FDH does so to a much lower extent than aqueous solution. Finally, a good agreement between experimental and theoretical kinetic isotope effects is found, thus giving some credit to our results.     

Wild-type and molten globular chorismate mutase achieve comparable catalytic rates using very different enthalpy/entropy compensations

The origin of the catalytic power of enzymes with a meta-stable native state,e.g.molten globular state,is an unsolved challenging issue in biochemistry.To help understand the possible differences between this special class of enzymes and the typical ones,we report here computer simulations of the catalysis of both the well-folded wild-type and the molten globular mutant of chorismate mutase.Using the ab initio quantum mechanical/molecular mechanical minimum free-energy path method,we determined the height of reaction barriers that are in good agreement with experimental measurements.Enzyme-substrate interactions were analyzed in detail to identify factors contributing to catalysis.Computed angular order parameters of backbone N–H bonds and side-chain methyl groups suggested site-specific,non-uniform rigidity changes of the enzymes during catalysis.The change of conformational entropy from the ground state to the transition state revealed distinctly contrasting entropy/enthalpy compensations in the dimeric wild-type enzyme and its molten globular monomeric variant.A unique catalytic strategy was suggested for enzymes that are natively molten globules:some may possess large conformational flexibility to provide strong electrostatic interactions to stabilize the transition state of the substrate and compensate for the entropy loss in the transition state.The equilibrium conformational dynamics in the reactant state were analyzed to quantify their contributions to the structural transitions enzymes needed to reach the transition states.The results suggest that large-scale conformational dynamics make important catalytic contributions to sampling conformational regions in favor of binding the transition state of substrate.     

Activation of orotidine 5'-monophosphate decarboxylase by phosphite dianion: the whole substrate is the sum of two parts

We report that the binding of phosphite dianion to orotidine 5'-monophosphate decarboxylase (OMPDC) results in an 80 000-fold increase in kcat/Km for decarboxylation of the truncated substrate, 1-(beta-d-erythrofuranosyl)orotic acid (EO), which lacks a 5'-phosphodianion moiety. The intrinsic binding energy (IBE) of phosphite dianion in the transition state is 7.8 kcal/mol, which represents a very large fraction of the 11.8 kcal/mol IBE of the phosphodianion group of the natural substrate orotidine 5'-monophosphate (OMP). The data give kcat = 160 +/- 70 s-1 for turnover of EO in the active site of OMPDC containing phosphite dianion, which is significantly larger than kcat = 15 s-1 for turnover of OMP. Despite the weaker binding of the individual EO and HPO32- "parts" (KmKd = 0.014 M2) than of OMP (Km = 1.6 x 10-6 M), once bound, OMPDC provides a slightly greater stabilization of the transition state for reaction of the parts than of the whole substrate. Thus, the covalent connection between the reacting portion of the substrate and the nonreacting phosphodianion group is not necessary for efficient catalysis. This implies that a major role of the phosphodianion group of OMP is to provide binding interactions that are used to drive an enzyme conformational change, resulting in formation of an active site environment optimized for transition state stabilization.     

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme

BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.     

Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy

Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450(cam) from Pseudomonas putida on fast time scales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450(cam) and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450(cam), three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450(cam) toward different substrates.     

Alpha/Beta-hydrolase fold enzymes: structures,functions and mechanisms

The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined. Relationships between active site anatomy, topology and conformational rearrangements in the protein molecule is discussed in the context of enzyme mechanism of action.