Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

Pathway of human AS3MT arsenic methylation

A synthetic gene encoding human As(III) S-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As(III). However, the C32S and C61S enzymes retained the ability to methylate MAs(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As(GS)3 binds to the enzyme much faster than inorganic As(III). Second, the major product of the first round of methylation is MAs(III), not MAs(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) Sci. Prog., 35, 396-416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183-191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates

Simulating Enzyme Catalysis

Chemistry is about transformations between compounds as the result of forming and breaking bonds between their atoms. A detailed knowledge of these processes should open the door to one of the most desired goals in this field, which is the control of the rate constants that govern the time dependence of the concentrations of reactants and products. Changes in the concentration and preparation of the reactants, the nature of the solvents and external conditions (such as pressure and temperature) can be employed to speed up or slow down a chemical reaction. One of the major breakthroughs in the field of chemical kinetics was achieved when it was recognized that certain substances, the catalysts , were able to accelerate chemical reactions without being consumed during the process

Spiers Memorial Lecture: Interplay of theory and computation in chemistry—examples from on-water organic catalysis, enzyme catalysis, and single-molecule fluctuations

In this lecture, several examples are considered that illustrate the interplay of experiment, theory, and computations. The examples include on-water catalysis of organic reactions, enzymatic catalysis, single molecule fluctuations, and some much earlier work on electron transfer and atom or group transfer reactions. Computations have made a major impact on our understanding and in the comparisons with experiments. There are also major advantages of analytical theories that may capture in a single equation an entire field and relate experiments of one type to those of another. Such a theory has a generic quality. These topics are explored in the present lecture

Investigation of Structural Dynamics of Enzymes and Protonation States of Substrates Using Computational Tools.

This review discusses the use of molecular modeling tools, together with existing experimental findings, to provide a complete atomic-level description of enzyme dynamics and function. We focus on functionally relevant conformational dynamics of enzymes and the protonation states of substrates. The conformational fluctuations of enzymes usually play a crucial role in substrate recognition and catalysis. Protein dynamics can be altered by a tiny change in a molecular system such as different protonation states of various intermediates or by a significant perturbation such as a ligand association. Here we review recent advances in applying atomistic molecular dynamics (MD) simulations to investigate allosteric and network regulation of tryptophan synthase (TRPS) and protonation states of its intermediates and catalysis. In addition, we review studies using quantum mechanics/molecular mechanics (QM/MM) methods to investigate the protonation states of catalytic residues of β-Ketoacyl ACP synthase I (KasA). We also discuss modeling of large-scale protein motions for HIV-1 protease with coarse-grained Brownian dynamics (BD) simulations

Principal basis for enzyme power

The reaction rate enhancement that enzymes produce had not been fully appreciated. The object of the article is to present the mechanism of the enormous catalytic power of the enzymes. I conclude that during substrate conversion to product the enzyme transfers firstly some additional small reactant group that must be initially presented in the active site of the enzyme to bound substrate (i) ; the enzyme regenerates during second substrate group transfer (ii); the active enzyme acts as a reactant of the enzymatic reaction (iii). The detailed chemical mechanisms of enzymatic reactions, such as a the well-studied reaction of the serine proteases family, the peptide bond hydrolysis catalyzed by α-chymotrypsin, and the glyceraldehyde-3-phosphate interconversion step in glycolysis, are in accordance with my conclusion

Local analysis of oxygen reduction catalysis by scanning vibrating electrode technique : a new approach to the study of biocorrosion

The scanning vibrating electrode technique (SVET)was employed to investigate oxygen reduction catalysis by the presence of enzyme in an aerobic medium. Heme protoporphyrin (hemin) was chosen as a model of the enzymes that are able to catalyze oxygen reduction. A strict experimental protocol was defined for preparing the graphite surface by deposition of hemin with a simple configuration mimicking the presence of enzyme on the samples. The same configuration was adapted to a stainless steel electrode. Different geometric arrangementswere investigated by SVET to approach the local conditions. The results demonstrated that hemin deposited on the electrode surface led to an increase in the cathodic current, which indicated a catalytic effect. Based on the SVET analysis, itwas demonstrated that hemin caused the appearance of galvanic cells on the material surface. The SVET proved able to locate active catalytic centres and therefore to foresee the contribution of the enzyme to the creation of galvanic cells, thus leading to localized corrosion. The application of SVET to the study of the interaction between biological molecules and material provides a newapproach for visualizing and understanding microbially influenced corrosion (MIC) in an aerobic medium