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 (pKe1 and pK32) and in the enzyme-substrate complexes (pKes1 and pKes2). The pKe1 of rhizopuspepsin (2.8) is near that of a normal carboxyl group and near the pKe1 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 pKe1 of pepsin (1.57) is thus abnormally low. The pKe2 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 pKes1 to 1.8 and the elevating of pKes2 to above 6. The pK alpha shifts due to substrate binding are much less pronounced in pepsin. Thus, the two enzyme-substrate complexes have similar pK alpha 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 alpha values. The major alteration caused by these mutations is the decrease in kcat values, while there is little change in Km. 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.
Lin
Y
Y
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City.
Fusek
M
M
Lin
X
X
Hartsuck
J A
JA
Kezdy
F J
FJ
Tang
J
J
D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D016333:HIV Protease / Q000378:metabolism; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010434:Pepsin A / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D009841:Oligonucleotides; D006859:Hydrogen; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease; D010434:Pepsin A; C035794:rhizopuspepsin