KINETIC MECHANISM OF THE INHIBITION OF HUMAN URINARY KALLIKREIN BY BASIC PANCREATIC TRYPSIN-INHIBITOR

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Ar g-Nan) at five different concentrations (10-20 mu M) by human urinary kallikrein was studied in the absence and in the presence of increasin g concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9. 15 nM). The data indicate that the inhibition of human urinary kallikr ein by BPTI is not a simple competitive inhibition as reported by othe rs, but that it is a competitive inhibition of the parabolic type, wit h two inhibitor molecules binding to one enzyme molecule, with the for mation of a ternary enzymatic complex. Statistical analysis of the exp erimental data supports the kinetic model proposed. The calculated val ues of the constants K-i and K-ii were 16.20 nM and 1.10 nM, respectiv ely. It is noteworthy that the K-ii < K-i, i.e., the second BPTI molec ule binds to the enzyme with a larger affinity suggesting that this se cond binding site was probably created or positively modulated as a co nsequence of the binding of the first BPTI molecule.