Abstract Volatile organic sulfur compounds (VOSC) are found in petroleum natural gas and condensa... more Abstract Volatile organic sulfur compounds (VOSC) are found in petroleum natural gas and condensates at trace levels. The abundance and δ34S values of VOSC were proposed as a proxy for thermal processes such as oil and gas generation during thermal maturation, thermochemical and microbial sulfate reduction, migration and expulsion. The understanding of VOSC formation and degradation mechanisms is needed to refine the use of δ34S values of VOSC as proxies for thermal processes over geological timescales in the subsurface. We therefore conducted pyrolysis experiments at 360 °C for 4, 12 and 72 h using pentane and H2S or pentanethiol as model compounds to study the formation and degradation pathways of VOSC and associated variations in δ34S values. The main products of these experiments are C1-C4 alkanes along with a variety of thiols and thiophenes, the most dominant VOSC formed. Most thiols were in equilibrium with H2S after 4 h based on their δ34S values. Thiophenes were first 34S depleted relative to H2S and only reached equilibrium at the 72 h experiment. The products and 34S fractionations in the pyrolysis experiment of pentanethiol at 360 °C and 12 h were similar to those of the experiment with H2S and pentane at the same conditions. This similarity suggests that pentanethiol is an intermediate product in the formation of aromatic VOSC during pyrolysis of pentane and H2S. Benzo- and dibenzothiophenes (BTs and DBTs) were formed in the liquid phase and their 34S depleted values relative to H2S indicated that they had not reached equilibrium. Ab-initio calculations of the thermodynamic properties of thiols, thiolanes, thiophenes BTs and DBTs were used to explain the relative abundance of products in the system and predict their abundance and S isotopic signature at reservoir conditions. The thermodynamic data suggests that at equilibrium, only small quantities of thiols and even smaller quantities of thiolanes can exist under our experimental conditions. Unlike thiols, the free energy of formation of thiophenes, BTs and DBTs is negative under these conditions indicating that the formation of these compounds is thermodynamically favored. The result suggests that the δ34S values of thiols are controlled by a very rapid equilibrium isotopic effect (EIE). On the other hand, aromatic sulfur compounds can preserve the δ34S value of the kinetic isotopic effect (KIE) associated with their formation for longer. However, under typical petroleum reservoir conditions, the formation of aromatic VOSC in the gas phase from short alkanes (
Proteins: Structure, Function, and Bioinformatics, 2009
Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the s... more Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the same time, these chemicals are highly toxic and can be used as nerve agents. G117H mutant of human Butyrylcholinesterase (BChE) was found to be capable of hydrolyzing certain OPs and protect against their toxicity. However, for therapeutic use, the rate of hydrolysis is too low. Its catalytic power can be improved by rational design, but the structure of the G117H mutant is first required. In this work, we determined, computationally, the three dimensional structure of the G117H BChE mutant. The structure was then validated by simulating acetylation of acetylthiocholine (ATC). Several plausible conformers of G117H BChE were examined but only the (62,−75) conformer fully reproduced catalytic effect. The (62,−75) conformer is, therefore, suggested as the structure adopted by the G117H BChE mutant. This conformer is shown to explain the loss of esterase activity observed for the G122H Acetyl...
The ability to design catalysis largely depends on our understanding of the electrostatic effect ... more The ability to design catalysis largely depends on our understanding of the electrostatic effect of the surrounding on the bonds participating in the reaction. Here, we used a simplistic model of point charges (PCs) to determine a set of rules guiding how to construct PC-bond arrangement that can strengthen or weaken different chemical bonds. Using valence bond theory to calculate the in situ bond energies, we show that the effect of the PC mainly depends on the bond’s dipole moment irrespective of its type (being covalent or charge shift). That is, polar bonds are getting stronger or weaker depending on the sign and location of the PC, whereas non- or weakly polar bonds become stronger or weaker depending only on the location of the PC and to a smaller extent compared with polar bonds. We also show that for polar bonds, the maximal bond strengthening and weakening effect can be achieved when the PC is placed along the bond axis, as close as possible to the more and less polarizable...
The attached data is the raw data used in the paper-"Sulfur isotopic composition of gas-phas... more The attached data is the raw data used in the paper-"Sulfur isotopic composition of gas-phase organic sulfur compounds provides insights into the thermal maturation of organic-rich rocks", accepted for publication in Geochimica et Cosmochimica Acta. In this work, we study the formation pathways of Volatile Organic Sulfur Compounds (VOSC), their specific sulfur isotope (d34S) values, and interaction with organic-rich sedimentary rock (or 'source rock') from the Ghareb Formation, a Type II-S source rock deposited in an upwelling marine environment. The source rock was exposed to laboratory controlled thermal maturation. We used a semi-open, non-isothermal pyrolysis system between 200 and 440 °C, and analyzed the molecular composition and compound specific d34S value of the evolved gases. For independent comparison, Quantum chemistry calculations (ab-initio) for S isotope exchange between H2S and representative VOSC were also included. All experimental and ab-intio calculated data is supplied in the attached file. First, the reader can find the first three tables provided in the published paper (Table 1-3). Afterward, additional data is provided, which in the text is referred as supplementary. Tables S1-S5 are the optimized coordinates for molecular structures obtained by ab-initio quantum calculations as described in the paper. Table S6 shows the vibrational temperatures obtained from the Gaussian 09 program and used to calculate the equilibrium constants for the equilibrium isotopic effect between the different organic sulfur compounds used in the study and co-existing H2S and given in table S7. Table S8 contains the amount of the major gas composition in the rock used for the experiment.
Ab initio classical valence bond theory in terms of localized orbitals has several advantages ove... more Ab initio classical valence bond theory in terms of localized orbitals has several advantages over electronic structure methods based on canonical delocalized Hartree-Fock molecular orbitals, with two key distinctions being greater applicability to inherently multiconfigurational systems (also called multireference systems or strongly correlated systems) and great interpretability in terms of covalent and ionic valence bond configurations (also called valence bond structures). This can be especially advantageous for applications to chemical reactions. However, until now only limited work tested the quantitative accuracy for the energetics of chemical reactions. The present study provides such tests and validations for a representative test set of six barrier heights corresponding to forward and reverse barriers of three hydrogen transfer reactions. In particular, we test the valence bond self-consistent-field theory (VBSCF) and three post-VBSCF methods that use VBSCF as a reference function for adding dynamic correlation, in particular valence bond configuration interaction (VBCI), breathing orbital valence bond (BOVB), and valence bond second-order perturbation theory (VBPT2). The VBSCF method itself is, as expected, not quantitatively accurate, with a mean unsigned error (MUE) for the six barrier heights of $17 kcal/mol. But the post-VBSCF methods are found to be quite successful. Depending on the basis set, and valence bond structure selection we obtain MUEs (in kcal/mol) as low as 3.7 for VBCI, 4.5 for BOVB, and (using a bigger basis-set) 1.3 for VBPT2. These compare well, on the same data set, with 1.6 for coupled clusters with singles and doubles (CCSD) and 1.5 for multireference second order perturbation theory based on a complete active space self-consistent field reference function (MRPT2). We discuss the results in terms of the pros and cons of each of these methods.
Publisher Summary This chapter focuses on the progress and the current state of structure/functio... more Publisher Summary This chapter focuses on the progress and the current state of structure/function correlations of biomolecules. One of the primary goals of modern molecular biology is to provide a reliable correlation between the structure and function of biomolecules. The chapter reviews the classical force field approaches and their use in macromolecular modeling—such as molecular mechanics, molecular dynamics, and evaluation of free energy using the free energy perturbation (FEP) and linear response approximation (LRA) approaches. It also emphasizes the need for proper long range treatments and the problem with using a single very long trajectory to explore biological functions. The chapter considers models for studies of electrostatic energies in proteins and their use in evaluation of pKa's, redox potentials, and binding free energies. The main part of the review deals with the modeling of enzyme catalysis. This part will consider several modeling approaches ranging from the empirical valence bond (EVB) to different molecular hybrid quantum mechanics/molecular mechanics (QM/MM) orbital approaches as well as approaches that attempt to treat the entire enzyme quantum mechanically. The potential and actual current performance of QM/MM and related approaches is presented. It is pointed out that while the EVB provides the most reliable results, alternative QM (MO)/MM methods are starting to provide reasonable results as well. There is no doubt about massive progress in the use of reliable QM/MM and related approaches. It is also certain that there will be a growing realization that consistent simulation studies provide the ultimate tool for understanding structure/function correlations in proteins.
Understanding enzyme catalysis and developing ability to control of it are two great challenges i... more Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational-based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild-type haloalkane dehalogenase (wt-DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non-optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate.
The valence bond (VB) method has enjoyed its prime time during the early stages in the field of q... more The valence bond (VB) method has enjoyed its prime time during the early stages in the field of quantum chemistry. After the advent of molecular orbital methods VB lost its popularity but continued to be improved and refined by a small community of scientists who appreciated its power of revealing insight into the origins of chemical reactivity. This review summarizes
Proteins: Structure, Function, and Bioinformatics, 2010
This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds b... more This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds by G117H-BChE. It is a theoretical study that focuses on the role of the G117H mutation in the dephosphorylation step. Various proposed mechanisms are examined. We show that His117 acts as a general base by activating a water molecule, and thus assisting its nucleophilic attack on the phosphate. The calculated reaction energy profile agrees well with the experimental data. Moreover, analysis of the reaction via its two hypothetical elementary steps, proton transfer and hydroxide attack, supports the role of His117 as a general base. Further support to the proposed mechanism is gained by structural comparison of the active site to RNAse A, which has similar composition of substrate and functional groups. The similarity between these enzymes extends beyond the structure and also becomes evident when comparing functionality of various active sites residues as well as rate-pH dependence obtained in the two cases. Moreover, it is demonstrated that an extended form of Bevilacqua's model (Biochemistry 2003;42:2259-2265) may resolve the apparent contradictions between the proposed mechanism and various experimental observations regarding rate-pH dependence. Finally, that same model is shown to rationalize the hydrolase activity of G117D BChE, an observation which is considered puzzling. It is concluded that G117H-BChE hydrolyzes echothiophate and possibly other OP compounds via a general acid-base mechanism. On the basis of this mechanism, one can now proceed with rational design aimed at improving the enzyme by exploiting both the structural and mechanistic knowledge.
The steric effect, exerted by enzymes on their reacting substrates, has been considered as a majo... more The steric effect, exerted by enzymes on their reacting substrates, has been considered as a major factor in enzyme catalysis. In particular, it has been proposed that enzymes catalyze their reactions by pushing their reacting fragments to a catalytic configuration which is sometimes called near attack configuration (NAC). This work uses computer simulation approaches to determine the relative importance of the steric contribution to enzyme catalysis. The steric proposal is expressed in terms of well defined thermodynamic cycles that compare the reaction in the enzyme to the corresponding reaction in water. The S N2 reaction of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, which was used in previous studies to support the strain concept is chosen as a test case for this proposal. The empirical valence bond (EVB) method provides the reaction potential surfaces in our studies. The reliability and efficiency of this method make it possible to obtain stable results for the steric free energy. Two independent strategies are used to evaluate the actual magnitude of the steric effect. The first applies restraints on the substrate coordinates in water in a way that mimics the steric effect of the protein active site. These restraints are then released and the free energy associated with the release process provides the desired estimate of the steric effect. The second approach eliminates the electrostatic interactions between the substrate and the surrounding in the enzyme and in water, and compares the corresponding reaction profiles. The difference between the resulting profiles provides a direct estimate of the nonelectrostatic contribution to catalysis and the corresponding steric effect. It is found that the nonelectrostatic contribution is about-0.7 kcal/mol while the full "apparent steric contribution" is about-2.2 kcal/mol. The apparent steric effect includes about-1.5 kcal/mol electrostatic contribution. The total electrostatic contribution is found to account for almost all the observed catalytic effect (∼-6.1 kcal/mol of the-6.8 calculated total catalytic effect). Thus, it is concluded that the steric effect is not the major source of the catalytic power of haloalkane dehalogenase. Furthermore, it is found that the largest component of the apparent steric effect is associated with the solvent reorganization energy. This solvent-induced effect is quite different from the traditional picture of balance between the repulsive interaction of the reactive fragments and the steric force of the protein.
The catalytic reaction of chorismate mutase (CM) has been the subject of major current attention.... more The catalytic reaction of chorismate mutase (CM) has been the subject of major current attention. Nevertheless, the origin of the catalytic power of CM remains an open question. In particular, it has not been clear whether the enzyme works by providing electrostatic transition state stabilization (TSS), by applying steric strain, or by populating near attack conformation (NAC). The present work explores this issue by a systematic quantitative analysis. The overall catalytic effect is reproduced by the empirical valence bond (EVB) method. In addition, the binding free energy of the ground state and the transition state is evaluated, demonstrating that the enzyme works by TSS. Furthermore, the evaluation of the electrostatic contribution to the reduction of the activation energy establishes that the TSS results from electrostatic effects. It is also found that the apparent NAC effect is not the reason for the catalytic effect but the result of the TSS. It is concluded that in CM as in other enzymes the key catalytic effect is electrostatic TSS. However, since the charge distribution of the transition state and the reactant state is similar, the stabilization of the transition state leads to reduction in the distance between the reacting atoms in the reactant state.
Abstract Volatile organic sulfur compounds (VOSC) are found in petroleum natural gas and condensa... more Abstract Volatile organic sulfur compounds (VOSC) are found in petroleum natural gas and condensates at trace levels. The abundance and δ34S values of VOSC were proposed as a proxy for thermal processes such as oil and gas generation during thermal maturation, thermochemical and microbial sulfate reduction, migration and expulsion. The understanding of VOSC formation and degradation mechanisms is needed to refine the use of δ34S values of VOSC as proxies for thermal processes over geological timescales in the subsurface. We therefore conducted pyrolysis experiments at 360 °C for 4, 12 and 72 h using pentane and H2S or pentanethiol as model compounds to study the formation and degradation pathways of VOSC and associated variations in δ34S values. The main products of these experiments are C1-C4 alkanes along with a variety of thiols and thiophenes, the most dominant VOSC formed. Most thiols were in equilibrium with H2S after 4 h based on their δ34S values. Thiophenes were first 34S depleted relative to H2S and only reached equilibrium at the 72 h experiment. The products and 34S fractionations in the pyrolysis experiment of pentanethiol at 360 °C and 12 h were similar to those of the experiment with H2S and pentane at the same conditions. This similarity suggests that pentanethiol is an intermediate product in the formation of aromatic VOSC during pyrolysis of pentane and H2S. Benzo- and dibenzothiophenes (BTs and DBTs) were formed in the liquid phase and their 34S depleted values relative to H2S indicated that they had not reached equilibrium. Ab-initio calculations of the thermodynamic properties of thiols, thiolanes, thiophenes BTs and DBTs were used to explain the relative abundance of products in the system and predict their abundance and S isotopic signature at reservoir conditions. The thermodynamic data suggests that at equilibrium, only small quantities of thiols and even smaller quantities of thiolanes can exist under our experimental conditions. Unlike thiols, the free energy of formation of thiophenes, BTs and DBTs is negative under these conditions indicating that the formation of these compounds is thermodynamically favored. The result suggests that the δ34S values of thiols are controlled by a very rapid equilibrium isotopic effect (EIE). On the other hand, aromatic sulfur compounds can preserve the δ34S value of the kinetic isotopic effect (KIE) associated with their formation for longer. However, under typical petroleum reservoir conditions, the formation of aromatic VOSC in the gas phase from short alkanes (
Proteins: Structure, Function, and Bioinformatics, 2009
Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the s... more Organophosphate ester (OP) compounds are known for their ubiquitous use as insecticides. At the same time, these chemicals are highly toxic and can be used as nerve agents. G117H mutant of human Butyrylcholinesterase (BChE) was found to be capable of hydrolyzing certain OPs and protect against their toxicity. However, for therapeutic use, the rate of hydrolysis is too low. Its catalytic power can be improved by rational design, but the structure of the G117H mutant is first required. In this work, we determined, computationally, the three dimensional structure of the G117H BChE mutant. The structure was then validated by simulating acetylation of acetylthiocholine (ATC). Several plausible conformers of G117H BChE were examined but only the (62,−75) conformer fully reproduced catalytic effect. The (62,−75) conformer is, therefore, suggested as the structure adopted by the G117H BChE mutant. This conformer is shown to explain the loss of esterase activity observed for the G122H Acetyl...
The ability to design catalysis largely depends on our understanding of the electrostatic effect ... more The ability to design catalysis largely depends on our understanding of the electrostatic effect of the surrounding on the bonds participating in the reaction. Here, we used a simplistic model of point charges (PCs) to determine a set of rules guiding how to construct PC-bond arrangement that can strengthen or weaken different chemical bonds. Using valence bond theory to calculate the in situ bond energies, we show that the effect of the PC mainly depends on the bond’s dipole moment irrespective of its type (being covalent or charge shift). That is, polar bonds are getting stronger or weaker depending on the sign and location of the PC, whereas non- or weakly polar bonds become stronger or weaker depending only on the location of the PC and to a smaller extent compared with polar bonds. We also show that for polar bonds, the maximal bond strengthening and weakening effect can be achieved when the PC is placed along the bond axis, as close as possible to the more and less polarizable...
The attached data is the raw data used in the paper-"Sulfur isotopic composition of gas-phas... more The attached data is the raw data used in the paper-"Sulfur isotopic composition of gas-phase organic sulfur compounds provides insights into the thermal maturation of organic-rich rocks", accepted for publication in Geochimica et Cosmochimica Acta. In this work, we study the formation pathways of Volatile Organic Sulfur Compounds (VOSC), their specific sulfur isotope (d34S) values, and interaction with organic-rich sedimentary rock (or 'source rock') from the Ghareb Formation, a Type II-S source rock deposited in an upwelling marine environment. The source rock was exposed to laboratory controlled thermal maturation. We used a semi-open, non-isothermal pyrolysis system between 200 and 440 °C, and analyzed the molecular composition and compound specific d34S value of the evolved gases. For independent comparison, Quantum chemistry calculations (ab-initio) for S isotope exchange between H2S and representative VOSC were also included. All experimental and ab-intio calculated data is supplied in the attached file. First, the reader can find the first three tables provided in the published paper (Table 1-3). Afterward, additional data is provided, which in the text is referred as supplementary. Tables S1-S5 are the optimized coordinates for molecular structures obtained by ab-initio quantum calculations as described in the paper. Table S6 shows the vibrational temperatures obtained from the Gaussian 09 program and used to calculate the equilibrium constants for the equilibrium isotopic effect between the different organic sulfur compounds used in the study and co-existing H2S and given in table S7. Table S8 contains the amount of the major gas composition in the rock used for the experiment.
Ab initio classical valence bond theory in terms of localized orbitals has several advantages ove... more Ab initio classical valence bond theory in terms of localized orbitals has several advantages over electronic structure methods based on canonical delocalized Hartree-Fock molecular orbitals, with two key distinctions being greater applicability to inherently multiconfigurational systems (also called multireference systems or strongly correlated systems) and great interpretability in terms of covalent and ionic valence bond configurations (also called valence bond structures). This can be especially advantageous for applications to chemical reactions. However, until now only limited work tested the quantitative accuracy for the energetics of chemical reactions. The present study provides such tests and validations for a representative test set of six barrier heights corresponding to forward and reverse barriers of three hydrogen transfer reactions. In particular, we test the valence bond self-consistent-field theory (VBSCF) and three post-VBSCF methods that use VBSCF as a reference function for adding dynamic correlation, in particular valence bond configuration interaction (VBCI), breathing orbital valence bond (BOVB), and valence bond second-order perturbation theory (VBPT2). The VBSCF method itself is, as expected, not quantitatively accurate, with a mean unsigned error (MUE) for the six barrier heights of $17 kcal/mol. But the post-VBSCF methods are found to be quite successful. Depending on the basis set, and valence bond structure selection we obtain MUEs (in kcal/mol) as low as 3.7 for VBCI, 4.5 for BOVB, and (using a bigger basis-set) 1.3 for VBPT2. These compare well, on the same data set, with 1.6 for coupled clusters with singles and doubles (CCSD) and 1.5 for multireference second order perturbation theory based on a complete active space self-consistent field reference function (MRPT2). We discuss the results in terms of the pros and cons of each of these methods.
Publisher Summary This chapter focuses on the progress and the current state of structure/functio... more Publisher Summary This chapter focuses on the progress and the current state of structure/function correlations of biomolecules. One of the primary goals of modern molecular biology is to provide a reliable correlation between the structure and function of biomolecules. The chapter reviews the classical force field approaches and their use in macromolecular modeling—such as molecular mechanics, molecular dynamics, and evaluation of free energy using the free energy perturbation (FEP) and linear response approximation (LRA) approaches. It also emphasizes the need for proper long range treatments and the problem with using a single very long trajectory to explore biological functions. The chapter considers models for studies of electrostatic energies in proteins and their use in evaluation of pKa's, redox potentials, and binding free energies. The main part of the review deals with the modeling of enzyme catalysis. This part will consider several modeling approaches ranging from the empirical valence bond (EVB) to different molecular hybrid quantum mechanics/molecular mechanics (QM/MM) orbital approaches as well as approaches that attempt to treat the entire enzyme quantum mechanically. The potential and actual current performance of QM/MM and related approaches is presented. It is pointed out that while the EVB provides the most reliable results, alternative QM (MO)/MM methods are starting to provide reasonable results as well. There is no doubt about massive progress in the use of reliable QM/MM and related approaches. It is also certain that there will be a growing realization that consistent simulation studies provide the ultimate tool for understanding structure/function correlations in proteins.
Understanding enzyme catalysis and developing ability to control of it are two great challenges i... more Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational-based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild-type haloalkane dehalogenase (wt-DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non-optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate.
The valence bond (VB) method has enjoyed its prime time during the early stages in the field of q... more The valence bond (VB) method has enjoyed its prime time during the early stages in the field of quantum chemistry. After the advent of molecular orbital methods VB lost its popularity but continued to be improved and refined by a small community of scientists who appreciated its power of revealing insight into the origins of chemical reactivity. This review summarizes
Proteins: Structure, Function, and Bioinformatics, 2010
This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds b... more This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds by G117H-BChE. It is a theoretical study that focuses on the role of the G117H mutation in the dephosphorylation step. Various proposed mechanisms are examined. We show that His117 acts as a general base by activating a water molecule, and thus assisting its nucleophilic attack on the phosphate. The calculated reaction energy profile agrees well with the experimental data. Moreover, analysis of the reaction via its two hypothetical elementary steps, proton transfer and hydroxide attack, supports the role of His117 as a general base. Further support to the proposed mechanism is gained by structural comparison of the active site to RNAse A, which has similar composition of substrate and functional groups. The similarity between these enzymes extends beyond the structure and also becomes evident when comparing functionality of various active sites residues as well as rate-pH dependence obtained in the two cases. Moreover, it is demonstrated that an extended form of Bevilacqua's model (Biochemistry 2003;42:2259-2265) may resolve the apparent contradictions between the proposed mechanism and various experimental observations regarding rate-pH dependence. Finally, that same model is shown to rationalize the hydrolase activity of G117D BChE, an observation which is considered puzzling. It is concluded that G117H-BChE hydrolyzes echothiophate and possibly other OP compounds via a general acid-base mechanism. On the basis of this mechanism, one can now proceed with rational design aimed at improving the enzyme by exploiting both the structural and mechanistic knowledge.
The steric effect, exerted by enzymes on their reacting substrates, has been considered as a majo... more The steric effect, exerted by enzymes on their reacting substrates, has been considered as a major factor in enzyme catalysis. In particular, it has been proposed that enzymes catalyze their reactions by pushing their reacting fragments to a catalytic configuration which is sometimes called near attack configuration (NAC). This work uses computer simulation approaches to determine the relative importance of the steric contribution to enzyme catalysis. The steric proposal is expressed in terms of well defined thermodynamic cycles that compare the reaction in the enzyme to the corresponding reaction in water. The S N2 reaction of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, which was used in previous studies to support the strain concept is chosen as a test case for this proposal. The empirical valence bond (EVB) method provides the reaction potential surfaces in our studies. The reliability and efficiency of this method make it possible to obtain stable results for the steric free energy. Two independent strategies are used to evaluate the actual magnitude of the steric effect. The first applies restraints on the substrate coordinates in water in a way that mimics the steric effect of the protein active site. These restraints are then released and the free energy associated with the release process provides the desired estimate of the steric effect. The second approach eliminates the electrostatic interactions between the substrate and the surrounding in the enzyme and in water, and compares the corresponding reaction profiles. The difference between the resulting profiles provides a direct estimate of the nonelectrostatic contribution to catalysis and the corresponding steric effect. It is found that the nonelectrostatic contribution is about-0.7 kcal/mol while the full "apparent steric contribution" is about-2.2 kcal/mol. The apparent steric effect includes about-1.5 kcal/mol electrostatic contribution. The total electrostatic contribution is found to account for almost all the observed catalytic effect (∼-6.1 kcal/mol of the-6.8 calculated total catalytic effect). Thus, it is concluded that the steric effect is not the major source of the catalytic power of haloalkane dehalogenase. Furthermore, it is found that the largest component of the apparent steric effect is associated with the solvent reorganization energy. This solvent-induced effect is quite different from the traditional picture of balance between the repulsive interaction of the reactive fragments and the steric force of the protein.
The catalytic reaction of chorismate mutase (CM) has been the subject of major current attention.... more The catalytic reaction of chorismate mutase (CM) has been the subject of major current attention. Nevertheless, the origin of the catalytic power of CM remains an open question. In particular, it has not been clear whether the enzyme works by providing electrostatic transition state stabilization (TSS), by applying steric strain, or by populating near attack conformation (NAC). The present work explores this issue by a systematic quantitative analysis. The overall catalytic effect is reproduced by the empirical valence bond (EVB) method. In addition, the binding free energy of the ground state and the transition state is evaluated, demonstrating that the enzyme works by TSS. Furthermore, the evaluation of the electrostatic contribution to the reduction of the activation energy establishes that the TSS results from electrostatic effects. It is also found that the apparent NAC effect is not the reason for the catalytic effect but the result of the TSS. It is concluded that in CM as in other enzymes the key catalytic effect is electrostatic TSS. However, since the charge distribution of the transition state and the reactant state is similar, the stabilization of the transition state leads to reduction in the distance between the reacting atoms in the reactant state.
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Papers by Avital Shurki