Enzyme Structure and Catalysis

The draggability of enzymes as targets reflects the evolution of enzyme structure to efficiently perform catalysis of chemical reactions, as discussed in the following section. 

Source Reprinted from G. Klebe, J. Mol. Biol. 237, p. 224 copyright 1994 with permission from Elsevier. 

The active site of DHFR illustrates several features that are common to enzyme active sites. Some of the salient features of active site structure that relate to enzyme catalysis and ligand (e.g., inhibitor) interactions have been enumerated by Copeland (2000)  

The active site of an enzyme is small relative to the total volume of the enzyme. 

The active site is three-dimensional—that is, amino acids and cofactors in the active site are held in a precise arrangement with respect to one another and with respect to the structure of the substrate molecule. This active site three-dimensional structure is formed as a result of the overall tertiary structure of the protein. 

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The instrumentation needed for solution studies of enzyme structure and catalysis at subzero temperatures is not difficult to build and operate. Detailed discussion of the technology is beyond the scope of this article, but a number of recent treatments provide all necessary information JJFink and Geeves, 1979 Douzou, 1974, 1977a,b). 

Dunn AR (2003) Sensitizer-linked substrates as probes of heme enzyme structure and catalysis. PhD, California Institute of Technology... 

T. C. Bruice and S. I Benkovic, Bioorganic Mechanisms, Vol. 1, W. A. Benjamin, New brk, 1966, pp. 1-258 W. P. Jencks, Catalysis in Chemistry and Enzymology, McGraw-Hill, New York, 1969 M. L. Bender, Mechanisms of Homogeneous Catalysis from Protons to Proteins, Wiley-Interscience, New York, 1971 C. Walsh, Enzymatic Reaction Mechanisms, W. H. Freeman, San Francisco, 1979 A. Fersht, Enzyme Structure and Mechanism, 2nd ed., W. H. Freeman, New York, 1985. 

In view of the arguments presented in this chapter, as well as in previous chapters, it seems that electrostatic effects are the most important factors in enzyme catalysis. Entropic factors might also be important in some cases but cannot contribute to the increase of kcJKM. Furthermore, as much as the correlation between structure and catalysis is concerned, it seems that the complimentarity between the electrostatic potential of the enzyme and the change in charges during the reaction will remain the best correlator. Finally, even in cases where the source of the catalytic activity of a given enzyme is hard to elucidate, it is expected that the methods presented in this book will provide the crucial ability to examine different hypothesis in a reliable way. 

Herein we provide a general overview of enzymatic catalysis in organic solvents, with a focus on the role of water and solvent on enzyme structure and function, and how an increased knowledge of this role has led to methods to activate enzymes for optimal use in organic media. 

Our purpose here is to review spectroscopic approaches, optical and vibrational, applied to the determination of enzyme structure and dynamics. We focus on hydride transfer reactions in protein catalysis. Vibrational spectroscopy is especially useful in the study of the molecular mechanism of enzymes because it is structurally specific and is of high resolution bond distortions as small as 0.01-0.001 A can be discerned by vibrational spectroscopy. It is at this level of atomic resolution that enzyme induced bond distortions usually manifest themselves. In addition, both enthalpic and entropic factors can be characterized by vibrational spectroscopy, sometimes in quantitative terms. Although most of the chapter is concerned with the structures of static protein-ligand complexes, the dynamics of how these complexes are formed and depleted has recently become a viable topic for scientific... 

Site-directed mutagenesis has become an important and widespread technique for the elucidation of structure-function relationships in proteins. However, the repercussions of mutations on both protein structure and catalysis are often subtle and, particularly in the case of mechanisms that require multiple catalytic steps, not always easily interpretable. Classical comparison of catalytic rate parameters between mutant and native enzymes where an amino acid substitution results in a change in the the rate-limiting step of a reaction are not necessarily valid (109). Thus, direct detection of reaction intermediates is an important means for assessing the effect of mutations on the mechanism and for accurately determining the role of various protein residues in catalysis. 

While developed as possible therapeutics, bisubstrate analogs have found great utility in the dissection and characterization of enzyme structure and mechanism. As discussed below, bisubstrate analogs have been used extensively in structural studies, where the use of natural substrates would result in catalysis, to investigate the architecture of the active site at the Michaelis complex, and to define structural changes at the active site produced by allosteric effectors. In some cases, bisubstrate analogs that are formed during the reaction (a type of mechanism-based inhibitor) can help to support or eliminate proposed chemical mechanisms. 

AR Fersht. Structure and Mechanism in Protein Science A Guide to Enzyme Catalysis and Protein Folding. New York WFl Freeman, 1999. 

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