REPROTONATION KINETICS OF ASPARTIC PROTEASES

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The purpose of this research is to document and characterize iso- mechanisms in aspartic proteases. The central features of the proposed iso-mechanism are partially rate-limiting rehydration and reprotonation of the aspartic carboxyl groups in the active site. Evidence from this laboratory in support of iso-mechanisms for aspartic proteases (and other enzymes) is diverse and includes: (a) solvent isotope effects on maximal velocities, (b) solvent isotope effects on onsets of inhibition, (c) solvent isotope effects on inhibition constants, (d) product inhibition kinetic patterns, (e) dead-end inhibition patterns, (f) progress curve analysis, and (g) modified and traditional induced transport kinetics of Britton (i.e., transpeptidation and isotopic exchange, respectively). Original experimental designs described in the current proposal include: (a) comparisons of rate constants and isotope effects on the recovery of pepsin from time-dependent inhibition by pepstatin incurred in the presence and absence of substrates, (b) steady-state kinetics of the initial inhibition of pepsin by pepstatin as a function of substrate concentration and identity, (c) effects of changes in the reactivity of water on iso-mechanism kinetics, (d) proton inventories on transpeptidation reactions, (e) kinetic competency for transpeptidation of peptides blocked at either the carboxyl or amino terminus, and (f) a search for burst phenomena in transient-state kinetics of peptide hydrolysis using highly sensitive pH indicator assays. The significance this proposal derives from the family of enzymes to which pepsin belongs, the aspartic proteases. Studies on pepsin serve as models to a series of clinically-significant enzymes, including most notably the HIV protease and renal renin. Similar isotope effects have been reported, suggesting that a rate-limiting reprotonation may be a common feature of the aspartic proteases. If true, then this kinetic mechanism will have an important relevance to the design of inhibitors. These should be designated to bind to the form of enzyme that is present in the greatest concentration in vivo, which this proposal holds is a form of free enzyme that has not yet undergone rehydration and reprotonation.