Thermolysin kinetic studies

K. Oyama, K. Kihara, and Y. Nonaka, On the mechanism of the action of thermolysin kinetic study of the thermolysin-catalyzed condensation reaction of N-benzyloxy-carbonyl-t-aspartic add with L-phenyl-alanine methyl ester, J. Chem. Soc. Perkin Trans. 2 1981a, 356-360. 

The preceding study is directly applicable to understanding the active site of thermolysin, since a recent kinetic study (6) comparing neutral protease from B. subtilis with thermolysin showed that the two enzymes have identical pH rate profiles that peak near pH 7. 

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

It is primarily for the above reasons that, in the view of this author, it is not yet possible to rmequivocably define the mechanistic role played by zinc ion for any zinc-enzyme. Nevertheless, with the exception of thermolysin, it is possible to arrive at reasonable mechanistic hypotheses for the various zinc enzymes considered in this review through the examination of data derived from both kinetic studies and from studies at equilibrium through judicious application of the anthropomorphic approach to the description of reaction mechanisms (50). 

Thermolysin belongs to a class of proteases (called neutral proteases) which are distinct from the serine proteases, sulfhydryl proteases, metal-loexopeptidases, and acid proteases. Neutral proteases A and B from Bacillus subtilis resemble thermolysin in molecular weight, substrate specificity, amino acid content, and metal ion dependence. Since physiological substrates are most likely proteins, it is difficult to design simple experiments that can be interpreted in terms of substrate specificity and relative velocities. Therefore, studies of substrate specificity and other kinetic parameters must be carried out on di- and tripeptides so that details of the mechanism of catalysis can be obtained and interpreted simply. 

Detailed information on the mechanism of biochemical reactions may be of crucial importance in designing new molecules having a pharmacological activity. For example, the detailed mechanism of protein hydrolysis by thermolysin has been studied at the QM/MM semiempirical level [25]. The various steps of the reaction and their transition states have been characterized. Fig. 3 (see color plate) shows the structure of the transition state of the rate-determining step. The important consequence of this approach is the fact that it is possible to evaluate the influence of the whole macro-molecular surroundings on the energetics of the process. It then becomes possible, for instance, to predict the influence of a mutation on the reaction kinetics. 

Using the reverse of the hydrolytic reaction, proteases are capable of catalyzing the formation of peptide bonds. Thermolysin is a powerful enzyme in catalyzing the synthesis of various useful oligopeptides, either in a free form or in an immobilized form in organic solvent media [58]. Recently, the systematic study about the stability of immobilized thermolysin has been reported [59]. In this study, the authors described the mechanism and kinetics for the inactivation of immobilized thermolysin with respect to the effects of organic solvents, kinds of support, water content, and so on. The strategy and results of their approach are discussed next. 

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