PH-DEPENDENCE OF KINETIC-PARAMETERS OF PEPSIN, RHIZOPUSPEPSIN, AND THEIR ACTIVE-SITE HYDROGEN-BOND MUTANTS

The pH dependence of the kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants has been determined. These data have permitted the calculation of two active-site ionization constants in the free enzymes (pK(e1) and pK(e2)) and in the enzyme-substrate complexes (pK(es1) and pK(es2)). The pk(e1) of rhizopuspepsin (2.8) is near that of a normal carboxyl group and near the pK(e1) of human immunodeficiency virus type 1 (HIV-1) protease (3.32) (Ido, E., Han, H. P., Kezdy, F. J., and Tang, J. (1991) J. Biol. Chem. 266, 24359-24366). The pK(e1) of pepsin (1.57) is thus abnormally low. The pK(e2) of rhizopuspepsin (4.44) is lower than that of pepsin (5.02) and HIV protease (6.80). The binding of substrate to rhizopuspepsin causes the lowering of pK(es1) to 1.8 and the elevating of pK(es2) to above 6. The pK(a) shifts due to substrate binding are much less pronounced in pepsin. Thus, the two enzyme-substrate complexes have similar pK(a) values. For both pepsin and rhizopuspepsin, the removal of hydrogen bonds to the active-site carboxyls by mutagenesis results in negligible changes in the four pK(a) values. The major alteration caused by these mutations is the decrease in k(cat) values, while there is little change in K(m). These observations suggest that these hydrogen bonds to the active-site aspartyls contribute little to the pH-activity relationships of the aspartic proteases. The role of the active-site hydrogen bonds may well be to preserve the conformational rigidity of the catalytic apparatus.