The purpose of this research is to test a new kinetic mechanism for porcine pepsin. The central feature of this mechanism is a rate-limiting reprotonation of the active site aspartic carboxyl groups, Asp-32 and Asp-215. Preliminary evidence in support of this mechanism include a solvent deuterium isotope effect on the maximal velocity, with none on V/K, when using a substrate whose V/K approaches diffusion control. Experiments designed to detect a rate-limiting reprotonation include: solvent deuterium isotope effects as a function of buffers of different concentration and pKa, product inhibition kinetics, steady-state isotopic exchange, and stopped-flow kinetics. A second important feature of the proposed mechanism is that the equilibrium for reprotonation lies far to the right; hence, the immediate form of enzyme after product release but before reprotonation is expected to be kinetically competent to synthesize peptide bonds. Experiments designed to detect synthetic competence involve back-exchange of labeled products into peptide substrates during catalytic turnover, but not from the products and free enzyme alone. 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 solvent isotope effects have been reported for renin, suggesting that a ratelimiting reprotonation may be a common feature of the aspartic proteases. If that is so, then this mechanism will have an important relevance to the design of inhibitors; these should be designed 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 reprotonation.
| Rebholz, K L; Northrop, D B (1994) Kinetics of enzymes with iso-mechanisms: dead-end inhibition of fumarase and carbonic anhydrase II. Arch Biochem Biophys 312:227-33 |
| Northrop, D B; Rebholz, K L (1994) Kinetics of enzymes with iso-mechanisms: analysis and display of progress curves. Anal Biochem 216:285-90 |
| Cho, Y K; Rebholz, K L; Northrop, D B (1994) Solvent isotope effects on the onset of inhibition of porcine pepsin by pepstatin. Biochemistry 33:9637-42 |
| Rebholz, K L; Northrop, D B (1993) Kinetics of enzymes with iso-mechanisms: analysis of product inhibition. Biochem J 296 ( Pt 2):355-60 |
