Biomedical subjects

P A Bash

Publications and source records attributed to P A Bash.

9 recordsLinked to original sources

PubMed1997

Simulation of the enzyme reaction mechanism of malate dehydrogenase.

A hybrid numerical method, which employs molecular mechanics to describe the bulk of the solvent-protein matrix and a semiempirical quantum-mechanical treatment for atoms near the reactive site, was utilized to simulate the minimum energy surface and reaction pathway for the interconversion of malate and oxaloacetate catalyzed by the enzyme malate dehydrogenase (MDH). A reaction mechanism for proton and hydride transfers associated with MDH and cofactor nicotinamide adenine dinucleotide (NAD) is deduced from the topology of the calculated energy surface. The proposed mechanism consists of (1) a sequential reaction with proton transfer preceding hydride transfer (malate to oxaloacetate direction), (2) the existence of two transition states with energy barriers of approximately 7 and 15 kcal/mol for the proton and hydride transfers, respectively, and (3) reactant (malate) and product (oxaloacetate) states that are nearly isoenergetic. Simulation analysis of the calculated energy profile shows that solvent effects due to the protein matrix dramatically alter the intrinsic reactivity of the functional groups involved in the MDH reaction, resulting in energetics similar to that found in aqueous solution. An energy decomposition analysis indicates that specific MDH residues (Arg-81, Arg-87, Asn-119, Asp-150, and Arg-153) in the vicinity of the substrate make significant energetic contributions to the stabilization of proton transfer and destabilization of hydride transfer. This suggests that these amino acids play an important role in the catalytic properties of MDH.

Binding Sites↗

PubMed1997

Computational enzymology.

Numerical simulations of enzyme reaction mechanisms are beginning to provide quantitative as well as qualitative insights. Methods based on hybrid quantum mechanical/molecular mechanical technique permit the natural inclusion of protein solvation effects. Coupled with modern experimental techniques, the numerical simulations are providing details at the atomic level about how enzyme structure influences its function.

Computer Simulation↗

PubMed1996

Combining Laue diffraction and molecular dynamics to study enzyme intermediates.

Two separate techniques, Laue diffraction and computational molecular dynamics (MD) simulations, have been independently developed to allow the visualization and assessment of transient structural states. Recent studies on isocitrate dehydrogenase show that computational MD simulations of an enzymatic Michaelis complex are consistent with difference Fourier electron density maps of the same structure from a Laue experiment. The use of independent MD studies during crystallographic refinement has allowed us to assign with confidence a number of additional contacts and features important for hydride transfer. We find that unrestrained independent MD simulations provides a very useful method of cross-validation for highly mobile atoms in regions of experimental density that are poorly defined. Likewise, information from Laue difference maps provides information about substrate conformation and interactions that greatly facilitate MD simulations.

Computer Simulation↗

PubMed1992

Simulation analysis of triose phosphate isomerase: conformational transition and catalysis.

A theoretical approach is employed to study the catalysis of the dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde 3-phosphate (GAP) reaction by the enzyme triose phosphate isomerase (TIM). The conformational change in a loop involved in protecting the active site from solvent is examined by use of X-ray data and molecular dynamics simulations. A mixed quantum-mechanics and molecular mechanics potential is used to determine the energy surface along the reaction path. The calculations address the role of the enzyme in lowering the barrier to reaction and provide a decomposition into specific residue contributions. To obtain a clearer understanding of the electronic effects, the polarization of the substrate carbonyl group by the active site residues is examined and compared with FTIR measurements on the wild-type and mutant forms of the enzyme.

Amino Acid Sequence↗

PubMed1991

Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analogue of the intermediate on the reaction pathway.

The glycolytic enzyme triosephosphate isomerase (TIM) catalyzes the interconversion of the three-carbon sugars dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP) at a rate limited by the diffusion of substrate to the enzyme. We have solved the three-dimensional structure of TIM complexed with a reactive intermediate analogue, phosphoglycolohydroxamate (PGH), at 1.9-A resolution and have refined the structure to an R-factor of 18%. Analysis of the refined structure reveals the geometry of the active-site residues and the interactions they make with the inhibitor and, by analogy, the substrates. The structure is consistent with an acid-base mechanism in which the carboxylate of Glu-165 abstracts a proton from carbon while His-95 donates a proton to oxygen to form an enediol (or enediolate) intermediate. The conformation of the bound substrate stereoelectronically favors proton transfer from substrate carbon to the syn orbital of Glu-165. The crystal structure suggests that His-95 is neutral rather than cationic in the ground state and therefore would have to function as an imidazole acid instead of the usual imidazolium. Lys-12 is oriented so as to polarize the substrate oxygens by hydrogen bonding and/or electrostatic interaction, providing stabilization for the charged transition state. Asn-10 may play a similar role.

Amino Acid Sequence↗

PubMed1991

Computer simulation and analysis of the reaction pathway of triosephosphate isomerase.

A theoretical approach designed for chemical reactions in the condensed phase is used to determine the energy along the reaction path of the enzyme triosephosphate isomerase. The calculations address the role of the enzyme in lowering the barrier to reaction and provide a decomposition into specific residue contributions. The results suggest that, although Lys-12 is most important, many other residues within 16 A of the substrate contribute and that histidine-95 as the imidazole/imidazolate pair could act as an acid/base catalyst.

Amino Acid Sequence↗

PubMed1987

Free energy calculations by computer simulation.

A fundamental problem in chemistry and biochemistry is understanding the role of solvation in determining molecular properties. Recent advances in statistical mechanical theory and molecular dynamics methodology can be used to solve this problem with the aid of supercomputers. By using these advances the free energies of solvation of all the chemical classes of amino acid side chains, four nucleic acid bases and other organic molecules can be calculated. The effect of a site-specific mutation on the stability of trypsin is predicted. The results are in good agreement with available experiments.

Amino Acids↗

PubMed1987

Calculation of the relative change in binding free energy of a protein-inhibitor complex.

By means of a thermodynamic perturbation method implemented with molecular dynamics, the relative free energy of binding was calculated for the enzyme thermolysin complexed with a pair of phosphonamidate and phosphonate ester inhibitors. The calculated difference in free energy of binding was 4.21 +/- 0.54 kilocalories per mole. This compares well with the experimental value of 4.1 kilocalories per mole. The method is general and can be used to determine a change or "mutation" in any system that can be suitably represented. It is likely to prove useful for protein and drug design.

Amides↗

PubMedDate not supplied

Free energy perturbation calculations on binding and catalysis after mutating Asn 155 in subtilisin.

Site-directed mutagenesis is a very powerful approach to altering the biological functions of proteins, the structural stability of proteins and the interactions of proteins with other molecules. Several experimental studies in recent years have been directed at estimating the changes in catalytic properties, (rates of binding and catalysis) in site-directed mutants of enzymes compared to the native enzymes. Simulation approaches to the study of complex molecules have also become more powerful, in no small measure owing to the increase in computer power. These simulations have often allowed results of experiments to be rationalized and understood mechanistically. A new approach called the free-energy pertubation method, which uses statistical mechanics and molecular dynamics can often be used for quantitative calculation of free energy differences. We have applied such a technique to calculate the differential free energy of binding and free energy of activation for catalysis of a tripeptide substrate by native subtilisin and a subtilisin mutant (Asn 155----Ala 155). Our studies lead to a calculated difference in free energy of binding which is relatively small, but a calculated change in free energy of catalysis which is substantial. These energies are very close to those determined experimentally (J. A. Wells and D. A. Estell, personal communication), which were not known to us until the simulations were completed. This demonstrates the predictive power and utility of theoretical simulation methods in studies of the effects of site-specific mutagenesis on both enzyme binding and catalysis.

Asparagine↗