A good understanding of how an enzyme conducts its catalytic affairs requires knowledge of the transition state structure associated with the highest energy barrier on the reaction progress curve. In order for catalysis to occur, this structure must be significantly more stable than the structure of the transition state for the corresponding uncatalyzed reaction, and it must also be significantly different from the structure of such enzyme-related stable states as unassociated enzyme and substrate in solution or enzyme-substrate complex. We have proposed a model for the acylation of two serine proteases, alpha-chymotrypsin and subtilisin Carlsberg, by substituted phenyl esters. Using this model, we have identified a number of substrates that will permit us to deduce reasonably complete transition state structures for enzyme-substrate association and attack of the enzymatic nucleophile on the substrate ester carbonyl. The main advantage of the proposed model is that is permits the transition state structures for the elementary steps of acylation to be determined without kinetic ambiguity. The methods used (and the transition state features) to be determined in this study will include: (1) proton inventories (hydrogenic reorganization processes); (2) Beta-deuterium secondary isotope effects (substrate heavy atom reorganizations); (3) activation parameters (enthalpic and entropic changes). The proposed studies are a step toward achieving the long range goal of this project, which is to identify those factors that contribute to the remarkable catalytic activity and selectivity of enzymes, with particular emphasis on proteases. Recognition of the importance of proteases in the complement system and in other important physiological processes such as blood clotting, reproductive fertilization, digestion, and protein turnover has revitalized interest in their structures and mechanism. Vertebrate chymotrypsin and bacterial subtilisin are of additional interest because they appear to represent an example of convergent evolution.
| Matta, M S; Andracki, M E (1988) Acylation of subtilisin A by aryl esters: contribution to rate limitation by a physical step preceding general acid-base catalysis. Biochemistry 27:8000-7 |
