The role of product release in enzyme catalysis.

Conceptual definitions of maximal velocity and the Michaelis constant are provided that do not involve the assumption of any rate-determining step. The experimental basis of those definitions is a combination of pre-steady state and steady state kinetic observations.     

Simultaneous catalysis and product separation by cross-linked enzyme crystals

Crystalline cross-linked xylose isomerase (CLXI, EC 5.3.1.5) and xylanase (CLX, EC 3.2.1.8) were studied in a packed-bed reactor for simultaneous catalytic reaction and separation of substrates from reaction products. Streptomyces rubiginosus xylose isomerase catalyzed a slow isomerization of L-arabinose to L-ribulose and an epimerization to L-ribose. In equilibrium the reaction mixture contained 52.5% arabinose, 22.5% ribulose, and 25% ribose. In a packed-bed column filled with CLXI, a simultaneous reaction and separation resulted in fractions where arabinose concentration varied between 100-0%, ribulose between 0-55%, and ribose between 0-100%. Trichoderma reesei xylanase II hydrolyzed and transferred xylotetraose mainly to xylotriose and xylobiose. In a packed-bed column filled with CLX, xylotetraose rapidly reacted to xylobiose and xylose by a mechanism that is not yet fully understood.     

Directed enzyme evolution and selections for catalysis based on product formation

Enzyme engineering by molecular modelling and site-directed mutagenesis can be remarkably efficient. Directed enzyme evolution appears as a more general strategy for the isolation of catalysts as it can be applied to most chemical reactions in aqueous solutions. Selections, as opposed to screening, allow the simultaneous analysis of protein properties for sets of up to about 10(14) different proteins. These approaches for the parallel processing of molecular information 'Is the protein a catalyst?' are reviewed here in the case of selections based on the formation of a specific reaction product. Several questions are addressed about in vivo and in vitro selections for catalysis reported in the literature. Can the selection system be extended to other types of enzymes? Does the selection control regio- and stereo-selectivity? Does the selection allow the isolation of enzymes with an efficient turnover? How should substrates be substituted or mimicked for the design of efficient selections while minimising the number of chemical synthesis steps? Engineering sections provide also some clues to design selections or to circumvent selection biases. A special emphasis is put on the comparison of in vivo and in vitro selections for catalysis.     

Enzymatic catalysis in nonaqueous solvents

Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.     

非水相体系酶催化反应研究进展

非水相酶催化反应是酶催化反应中的一个重要方面。非水相溶剂通常可增加底物溶解度,减少水相中的副反应,加快生物催化的速率和效率,在药物及药物中间体和食品等方面具有较大的应用价值。以下探讨了非水相体系对酶活力及酶促反应速率的影响因素,并阐述酶的化学修饰、固定化及定点突变对酶活力的影响,进一步分析无溶剂系统、反胶束、超临界流体及离子液体的不同溶剂体系对酶反应速率及催化效率的影响。此外,还列举一些非水相酶催化反应的应用实例。     

Enzymatic catalysis in a supercritical fluid

The enzyme alkaline phosphatase, EC 3. 1. 3. 1, was found to be active in a supercritical carbo dioxide solvent system. A batch reaction of disodium p-nitrophenyl phosphate with a 0.1 vol. % water solution in supercritical CO₂ at 100 atm and 35°C produced p-nitrophenol when catalyzed by alkaline phosphatase.     

Enzymatic catalysis in anhydrous organic solvents

Not only do enzymes work vigorously in anhydrous organic media, but in this unnatural milieu they acquire remarkable properties such as greatly enhanced stability, radically altered substrate and enantiomeric specificities, molecular memory, and the ability to catalyse unusual reactions.     

Allostery in enzyme catalysis

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Femtochemistry in enzyme catalysis: DNA photolyase

Photolyase uses light energy to split UV-induced cyclobutane pyrimidine dimers in damaged DNA. This photoenzyme encompasses a series of elementary dynamical processes during repair function from early photoinitiation by a photoantenna molecule to enhance repair efficiency, to in vitro photoreduction through aromatic residues to reconvert the cofactor to the active form, and to final photorepair to fix damaged DNA. The corresponding series of dynamics include resonance energy transfer, intraprotein electron transfer, and intermolecular electron transfer, bond breaking-making rearrangements and back electron return, respectively. We review here our recent direct studies of these dynamical processes in real time, which showed that all these elementary reactions in the enzyme occur within subnanosecond timescale. Active-site solvation was observed to play a critical role in the continuous modulation of catalytic reactions. As a model system for enzyme catalysis, we isolated the enzyme–substrate complex in the transition-state region and mapped out the entire evolution of unmasked catalytic reactions of DNA repair. These observed synergistic motions in the active site reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.     

Age-related effects in enzyme catalysis

Summary Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue.Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in old glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of young glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native old glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.Abbreviations CPL circular polarization of luminescence - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - GPDH D-glyceraldehyde-3-phosphate dehydrogenase - ENAD⁺ nicotinamide 1,N⁶-ethenoadenine dinucleotide     

Proton tunneling and enzyme catalysis

Summary It is proposed in this paper that enzymes, by virtue of a number of correctly positioned sites of interaction with substrates, can force the compression of hydrogen bonds, increasing the probability of proton transfer by quantum mechanical tunneling. By such a catalytic mechanism a rate enhancement of many orders of magnitude may be obtained with a very low energy input requirement. The mechanism would, however, require a highly structured catalyst.Pertinent aspects of hydrogen bond theory and of tunneling theory are briefly reviewed.Work supported by NIGMS Training Grant No. GM 678-07.     

Ribonuclease structure and catalysis: effects of crystalline enzyme, alcohol cryosolvents, low temperatures, and product inhibition

In order to determine the necessary conditions to stabilize intermediates in ribonuclease A catalysis at subzero temperatures for structural studies, we have examined the suitability of alcohol-based cryosolvents. On the basis of thermal denaturation transition curves, the enzyme is in the native conformation in high concentrations of ethanol and methanol, provided the temperature is suitably low. The effects of methanol on the catalytic properties for the hydrolysis for mono- and dinucleotide substrates also are consistent with the absence of adverse effects of the cosolvent. Significant methanolysis occurs in the presence of methanol as cosolvent. The kinetics of 2',3'-CMP hydrolysis are complicated by severe competitive product inhibition, both in aqueous and in methanolic solvents, accounting for the previously observed effect of substrate concentration on the observed Km. Computer-aided analysis allowed the determination of the inhibition constant as a function of experimental parameters. The reaction of ribonuclease A with 2',3'-CMP was investigated at subzero temperatures. The turnover reaction could be made negligible at temperatures below -60 degrees C at pH 3-6 in 70% methanol and below -35 degrees C at pH 2.1. The rate of the catalytic reaction with crystalline enzyme was compared to that of enzyme in solution for both 2',3'-CMP and the dinucleotide CpC. The rates were 50- and 200-fold slower, respectively, in the crystal. These investigations allowed calculation of the necessary conditions for NMR and X-ray diffraction experiments on the trapped enzyme--substrate intermediate.     

Enzymatic mechanisms for catalysis of enolization: ketosteroid isomerase

Breaking a carbon-hydrogen bond adjacent to a carbonyl is a slow step in a large number of chemical reactions. However, many enzymes are capable of catalyzing this reaction with great efficiency. One of the most proficient of these enzymes is 3-oxo-Delta5-steroid isomerase (KSI), which catalyzes the isomerization of a wide variety of 3-oxo-Delta5-steroids to their Delta4-conjugated isomers. In this review, the mechanism of KSI is discussed, with particular emphasis on energetic considerations. Both experimental and theoretical approaches are considered to explain the mechanistic details of the reaction.     

Protein dynamics and enzyme catalysis: insights from simulations

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Multiple conformational changes in enzyme catalysis

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.