The chemistry of enzymes

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Page, M. I. (ed.) (1984). The Chemistry of Enzyme Action. Elsevier, Amsterdam... 

Walsh, C. (1979). Enzymatic Reaction Mechanisms. W. H. Freeman, San Francisco Williams, A. (1984). In The Chemistry of Enzyme Action (ed. M. I. Page), Chap. 5. Elsevier, Amsterdam... 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

Goldstein, L., Manecke, G. The Chemistry of Enzyme ImmobiUzation, in Immobilized Enzyme Principles, (ed. Wingard, L. B., Katchalski-Katzin, E., Goldstein, L.), Academic Press, New York 1976... 

Figure 10. Structures of electron-transfer probes used to examine the chemistry of enzyme active sites which are deeply buried within the protein, in particular the cytochrome P450 [354].

Walt DR and Agayn VI. The chemistry of enzyme and protein immobilization with glutaraldehyde. Trends Anal. Chem. 1994 13 425 30. 

Anfinsen, Christian B. (1916-1991). An American biochemist who won the Nobel Prize for chemistry in 1972. His work involved the molecular basis of evolution and the chemistry of enzymes. He worked with Moore and Stein. His doctorate was granted from Harvard. 

M. J. Page, The chemistry of enzyme action, Elsevier, Amsterdam, 1984. 

The most serious limitation of stopped-flow methods is that one does not always have an optical signal for the reaction of interest and the optical signals cannot be interpreted rigorously if the extinction coefficients of intermediates or products are not known. For example, an enzyme intermediate may have an unknown extinction coefficient, and without an absolute measurement of concentration of the intermediate, one cannot obtain a unique solution to its rate of formation and decay (see below). For these reasons, a direct measurement of the conversion of substrate to product is required. Chemical-quench-flow methods allow such direct measurement of the chemistry of enzyme-catalyzed reactions and can be performed for nearly any reaction. One must recognize that these experiments are based on examining the enzyme as a stoichiometric reactant such that the concentration of enzyme required will depend upon the kinetics of the reaction and the sensitivity of the methods for detection of intermediates or products. Nonetheless, quench-flow experiments can be performed using as little as 20 fd of solution and a complete enzyme pathway can be solved using only... 

This study has important lessons for enzyme kinetic analysis. The use of pH variation and examination of isotope elfects can be a powerful combination to explore the chemistry of enzyme-catalyzed reactions and to dissect the contributions of individual reaction steps to the net steady-state turnover (27). Examination of the effects of pH on each step of the reaction pathway could resolve the contributions of ionizable groups toward ground-state binding energy and transition-state stabilization. The use of isotope effects by transient-state kinetic methods is more limited than in the steady state due to the errors involved in comparing two rate measurements. In the steady state, the ratio method has allowed isotope effects of less than 1% to be measured accurately (8a, 58). By transient-state kinetics, one would require at least a 10-20% change in rate to demonstrate a convincing difference between two rate measurements in most instances. 

During the long years of natural selection a particularly precise structural correspondence among apoenzymes and the substituents has been established in the chemistry of enzymes. The molecular adsorption... 

Michael /. Page (Ed.), The Chemistry of Enzyme Action 1984 Elsevier Science Publishers B. V. 

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