Anatomy of acetylcholinesterase catalysis: reaction dynamics analogy for human erythrocyte and electric eel enzymes

The anatomy of catalysis (i.e., reaction dynamics, thermodynamics and transition state structures) is compared herein for acetylcholinesterases from human erythrocytes and Electrophorus electricus. The two enzymes have similar relative activities for the substrate o-nitrochloroacetanilide and o-nitrophenyl acetate. In addition, with each substrate K values and solvent deuterium kinetic isotope effects for kES and kE are similar for the two enzymes. Solvent isotope effects in mixed isotopic buffers indicate that the acylation stages of o-nitrochloroacetanilide turnover by the two enzymes are rate-limited by virtual transition states that are weighted averages of contributions from transition states of serial chemical and physical steps. Similar experiments show that the transition states for Vmax of o-nitrophenyl acetate turnover by the two enzymes are stabilized by simple general acid-base (i.e., one-proton) catalysis. These comparisons demonstrate that acetylcholinesterases from diverse sources display functional analogy in that reaction dynamics and transition state structures are closely similar.     

Structure of histone acetyltransferases

Histone acetyltranferase (HAT) enzymes are the catalytic subunits of multisubunit protein complexes that acetylate specific lysine residues on the N-terminal regions of the histone components of chromatin to promote gene activation. These enzymes, which now include more than 20 members, fall into distinct families that generally have high sequence similarity and related substrate specificity within families, but have divergent sequence and substrate specificity between families. Significant insights into the mode of catalysis and histone substrate binding have been provided by the structure determination of the divergent HAT enzymes Hat1, Gcn5/PCAF and Esa1. A comparison of these structures reveals a structurally conserved central core domain that mediates extensive interactions with the acetyl-coenzyme A cofactor, and structurally divergent N and C-terminal domains. A correlation of these structures with other studies reveals that the core domain plays a particularly important role in histone substrate catalysis and that the N and C-terminal domains play important roles in histone substrate binding. These correlations imply a related mode of catalysis and histone substrate binding by a diverse group of HAT enzymes.     

Substrate ground state binding energy concentration is realized as transition state stabilization in physiological enzyme catalysis

Previously published kinetic data on the interactions of seventeen different enzymes with their physiological substrates are re-examined in order to understand the connection between ground state binding energy and transition state stabilization of the enzyme-catalyzed reactions. When the substrate ground state binding energies are normalized by the substrate molar volumes, binding of the substrate to the enzyme active site may be thought of as an energy concentration interaction; that is, binding of the substrate ground state brings in a certain concentration of energy. When kinetic data of the enzyme/substrate interactions are analyzed from this point of view, the following relationships are discovered: 1) smaller substrates possess more binding energy concentrations than do larger substrates with the effect dropping off exponentially, 2) larger enzymes (relative to substrate size) bind both the ground and transition states more tightly than smaller enzymes, and 3) high substrate ground state binding energy concentration is associated with greater reaction transition state stabilization. It is proposed that these observations are inconsistent with the conventional (Haldane) view of enzyme catalysis and are better reconciled with the shifting specificity model for enzyme catalysis.     

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.     

Systematic optimization model and algorithm for binding sequence selection in computational enzyme design

A systematic optimization model for binding sequence selection in computational enzyme design was developed based on the transition state theory of enzyme catalysis and graph‐theoretical modeling. The saddle point on the free energy surface of the reaction system was represented by catalytic geometrical constraints, and the binding energy between the active site and transition state was minimized to reduce the activation energy barrier. The resulting hyperscale combinatorial optimization problem was tackled using a novel heuristic global optimization algorithm, which was inspired and tested by the protein core sequence selection problem. The sequence recapitulation tests on native active sites for two enzyme catalyzed hydrolytic reactions were applied to evaluate the predictive power of the design methodology. The results of the calculation show that most of the native binding sites can be successfully identified if the catalytic geometrical constraints and the structural motifs of the substrate are taken into account. Reliably predicting active site sequences may have significant implications for the creation of novel enzymes that are capable of catalyzing targeted chemical reactions.     

Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity?

Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well-defined structure. This view of the transition-state ensemble is consistent with the nature of the protein molecule, as embodied and depicted in the protein energy landscape of folding, and binding, funnels.     

Catalytic site forms and controls in ATP synthase catalysis

A suggested minimal scheme for substrate binding by and interconversion of three forms of the catalytic sites of the ATP synthase is presented. Each binding change, that drives simultaneous interchange of the three catalytic site forms, requires a 120 degrees rotation of the gamma with respect to the beta subunits. The binding of substrate(s) at two catalytic sites is regarded as sufficing for near maximal catalytic rates to be attained. Although three sites do not need to be filled for rapid catalysis, during rapid bisite catalysis some enzyme may be transiently present with three sites filled. Forms with preferential binding for ADP and P(i) or for ATP are considered to arise from the transition state and participate in other steps of the catalysis. Intermediate forms and steps that may be involved are evaluated. Experimental evidence for energy-dependent steps and for control of coupling to proton translocation and transition state forms are reviewed. Impact of relevant past data on present understanding of catalytic events is considered. In synthesis a key step is suggested in which proton translocation begins to deform an open site so as to increase the affinity for ADP and P(i), that then bind and pass through the transition state, and yield tightly bound ATP in one binding change. ADP binding appears to be a key parameter controlling rotation during synthesis. In hydrolysis ATP binding to a loose site likely precedes any proton translocation, with proton movement occurring as the tight site form develops. Aspects needing further study are noted. Characteristics of the related MgADP inhibition of the F(1) ATPases that have undermined many observations are summarized, and relations of three-site filling to catalysis are assessed.     

Computational design of a lipase for catalysis of the Diels-Alder reaction

Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like nearattack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the α,β-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates (v _cat /v _uncat ) of up to 103 are predicted.     

Intrinsic effects of solvent polarity on enzymic activation energies

The effect of organic solvents on subtilisin Carlsberg catalysis has been investigated with the aid of a thermodynamic analysis. Saturation solubility experiments were performed to provide a quantitative measure of substrate desolvation from the reaction medium. This enabled calculation of the intrinsic enzymic activation energy and resulted in a linear free energy relationship with respect to solvent polarity. The results indicate that the intrinsic activation energy of subtilisin catalysis is lowest in polar organic solvents, which may be due to transition state stabilization of the enzyme's polar transition state for transesterification.     

Analysis of conserved residues of the human puromycin-sensitive aminopeptidase

The puromycin-sensitive aminopeptidase (ApPS) is a zinc metallopeptidase involved in the degradation of neuropeptides. Putative catalytic residues of the enzyme, Cys146, Glu338, and Lys396 were mutated, and the resultant mutant enzymes characterized. ApPS C146S exhibited normal catalytic activity, ApPS E338A exhibited decreased substrate binding, and ApPS K396I exhibited decreases in both substrate binding and catalysis. ApPS K396I and ApPS Y394F were analyzed with respect to transition state inhibitor binding. No effect was seen with the K396I mutation, but ApPS Y394F exhibited a 3.3-fold lower affinity for RB-3014, a transition state inhibitor, indicating that Tyr394 is involved in transition state stabilization.     

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L----D and D----L directions for all three enzymes to assess the degree to which abstraction of the alpha-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of alpha-3H from substrate to product and solvent exchange/substrate conversion experiments in 3H2O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.     

Ligand-protein interaction in biomembrane carriers. The induced transition fit of transport catalysis

Carrier-linked transport through biomembranes is treated under the view of catalysis. As in enzymes, substrate-protein interaction yields catalytic energy in overcoming the activation barrier. At variance with enzymes, catalytic energy is concentrated on structural changes of the carrier rather than on the substrate destabilization for facilitating the global protein rearrangements during transport. A transition state is invoked in which the binding site assumes the best fit to the substrate, whereas in the two ground (internal and external) states, the fit is poor. The maximum binding energy released in the transition state provides catalytic energy to enable the large carrier protein transformations associated with transport. This "induced transition fit" (ITF) of carrier catalysis provides a framework of rules, concerning specificity, unidirectional versus exchange type transport, directing inhibitors to the ground state instead of the transition state, and excluding simultaneous chemical and transport catalysis (vectorial group translocation). The possible role of external energy sources (ATP and Deltapsi) in supplementing the catalytic energy is elucidated. The analysis of the structure-function relationship based on new carrier structures may be challenged to account for the workings of the ITF.     

Characterization of the amino acids from Neisseria meningitidis methionine sulfoxide reductase B involved in the chemical catalysis and substrate specificity of the reductase step

Methionine sulfoxide reductases (Msrs) are antioxidant repair enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The Msr family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the S and R isomers of the sulfoxide function, respectively. Both classes of Msr share a similar three-step chemical mechanism involving first a reductase step that leads to the formation of a sulfenic acid intermediate. In this study, the invariant amino acids of Neisseria meningitidis MsrB involved in the reductase step catalysis and in substrate binding have been characterized by the structure-function relationship approach. Altogether the results show the following: 1) formation of the MsrB-substrate complex leads to an activation of the catalytic Cys-117 characterized by a decreased pKapp of approximately 2.7 pH units; 2) the catalytic active MsrB form is the Cys-117-/His-103+ species with a pKapp of 6.6 and 8.3, respectively; 3) His-103 and to a lesser extent His-100, Asn-119, and Thr-26 (via a water molecule) participate in the stabilization of the polarized form of the sulfoxide function and of the transition state; and 4) Trp-65 is essential for the catalytic efficiency of the reductase step by optimizing the position of the substrate in the active site. A scenario for the reductase step is proposed and discussed in comparison with that of MsrA.     

High pressure enhancement of enzymes: A review

While most current applications of high pressure (HP) are for inactivating deleterious enzymes, there is evidence that high pressure can induce stabilization and activation of some enzymes. Various other strategies have been employed to enhance enzyme stability, including; genetic engineering, immobilization, and operating in non-aqueous media. While each of these strategies has provided varying degrees of stability or activity enhancement, the application of high pressure may be a complementary, synergistic, or an additive enzyme enhancement technique. Over 25 enzymes that have exhibited high pressure stabilization and/or activation were compiled. Each enzyme discussed responds differently to high pressure depending on the pressure range, temperature, source, solvent or media, and substrate. Possible mechanisms for pressure-induced stabilization and activation are discussed and compared with current enzyme enhancement techniques. The compiled evidence of high pressure enzyme enhancement in this review indicates that pressure is an effective reaction parameter with potential for greater utilization in enzyme catalysis.     

Pyruvate site of pyruvate phosphate dikinase: crystal structure of the enzyme-phosphonopyruvate complex, and mutant analysis.

Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (K(i) = 3 microM), have been obtained in the presence of Mg(2+). The structure has been determined and refined at 2.2 A resolution, revealing that the Mg(2+)-bound phosphonopyruvate binds in the alpha/beta-barrel's central channel, at the C-termini of the beta-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK-phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg(2+)-bound phospholactate and the other with Mg(2+)-oxalate and ATP, revealed that the two enzymes share some key features that facilitate common modes of substrate binding. There are also important structural differences; most notably, the machinery for acid/base catalysis is different.     

Lipase-catalyzed transesterification in organic media: solvent effects on equilibrium and individual rate constants

The kinetics of the immobilized lipase B from Candida antarctica have been studied in organic solvents. This enzyme has been shown to be slightly affected by the water content of the organic media, and it does not seem to be subject to mass transfer limitations. On the other hand, some evidence indicates that the catalytic mechanism of reactions catalyzed by this lipase proceeds through the acyl-enzyme intermediate. Moreover, despite the fact that the immobilization support dramatically enhances the catalytic power of the enzyme, it does not interfere with the intrinsic solvent effect. Consequently, this enzyme preparation becomes optimum for studying the role played by the organic solvent in catalysis. To this end, we have measured the acylation and deacylation individual rate constants, and the binding equilibrium constant for the ester, in several organic environments. Data obtained show that the major effect of the organic solvent is on substrate binding, and that the catalytic steps are almost unaffected by the solvent, indicating the desolvation of the transition state. However, the strong decrease in binding for hydrophilic solvents such as THF and dioxane, compared to the rest of solvents, cannot be easily explained by means of thermodynamic arguments (desolvation of the ester substrate). For this reason, data have been considered as an indication of the existence of an unknown step in the catalytic pathway occurring prior to formation of the acyl-enzyme intermediate.     

Molecular mechanism of ADP-ribose hydrolysis by human NUDT5 from structural and kinetic studies

Human NUDT5 (hNUDT5) is an ADP-ribose (ADPR) pyrophosphatase (ADPRase) that plays important roles in controlling the intracellular levels of ADPR and preventing non-enzymatic ADP-ribosylation of proteins by hydrolyzing ADPR to AMP and ribose 5′-phosphate. We report the crystal structure of hNUDT5 in complex with a non-hydrolyzable ADPR analogue, α,β-methyleneadenosine diphosphoribose, and three Mg^2 + ions representing the transition state of the enzyme during catalysis. Analysis of this structure and comparison with previously reported hNUDT5 structures identify key residues involved in substrate binding and catalysis. In the transition-state structure, three metal ions are bound at the active site and are coordinated by surrounding residues and water molecules. A conserved water molecule is at an ideal position for nucleophilic attack on the α-phosphate of ADPR. The side chain of Glu166 on loop L9 changes its conformation to interact with the conserved water molecule compared with that in the substrate-bound structure and appears to function as a catalytic base. Mutagenesis and kinetic studies show that Trp28 and Trp46 are important for the substrate binding; Arg51 is involved in both the substrate binding and the catalysis; and Glu112 and Glu116 of the Nudix motif, Glu166 on loop L9, and Arg111 are critical for the catalysis. The structural and biochemical data together reveal the molecular basis of the catalytic mechanism of ADPR hydrolysis by hNUDT5. Specifically, Glu166 functions as a catalytic base to deprotonate a conserved water molecule that acts as a nucleophile to attack the α-phosphate of ADPR, and three Mg^2 + ions are involved in the activation of the nucleophile and the binding of the substrate. Structural comparison of different ADPRases also suggests that most dimeric ADPRases may share a similar catalytic mechanism of ADPR hydrolysis.     

Conformational aspects of inhibitor design: enzyme-substrate interactions in the transition state.

The theory of absolute reaction rates suggests that enzymes, like other catalysts, can enhance the rate of a reaction only to the extent that they bind the altered substrate in the transition state (S++) more tightly than they bind the substrate in the ground state (S). ES dissociation constants commonly fall in the physiological range, but recent kinetic studies indicate that formal ES++ dissociation constants of less than 10(-20) M are achieved by enzymes of several classes. Studies with stable analogues suggest that these remarkable powers of discrimination involve a tendency of the enzyme to close around S++ in such a way as to maximize binding contacts; that several parts of the substrate contribute to S++ binding; and that their contributions to binding affinity can be strongly synergistic.     

Rieske business: structure-function of Rieske non-heme oxygenases

Rieske non-heme iron oxygenases (RO) catalyze stereo- and regiospecific reactions. Recently, an explosion of structural information on this class of enzymes has occurred in the literature. ROs are two/three component systems: a reductase component that obtains electrons from NAD(P)H, often a Rieske ferredoxin component that shuttles the electrons and an oxygenase component that performs catalysis. The oxygenase component structures have all shown to be of the alpha3 or alpha3beta3 types. The transfer of electrons happens from the Rieske center to the mononuclear iron of the neighboring subunit via a conserved aspartate, which is shown to be involved in gating electron transport. Molecular oxygen has been shown to bind side-on in naphthalene dioxygenase and a concerted mechanism of oxygen activation and hydroxylation of the ring has been proposed. The orientation of binding of the substrate to the enzyme is hypothesized to control the substrate selectivity and regio-specificity of product formation.