Hydrolysis of D-valyl-L-leucyl-L-arginine 4-nitroanilide (120-640 M) by rat tissue kallikrein (rK1) (3.15 nM) was studied in both the absence and the presence of increasing concentrations of aprotinin (10.4-34.6 nM), a serine protease inhibitor also known as basic pancreatic trypsin inhibitor, which inhibits trypsin, chymotrypsin, plasmin and kallikrein. The data indicate that the inhibition of rK1 by aprotinin is a parabolic competitive inhibition, with two inhibitor molecules binding to one enzyme molecule. Statistical analysis of the data supports the kinetic model proposed. The calculated values of the constants Ki and Kii (mean + SE) were 26.4 ± 5.4 nM and 16.9 ± 11.4 nM, respectively. Ki ≈ Kii, suggests that the aprotinin molecules bind to two enzyme sites with approximately the same affinity. Parabolic competitive inhibition was also reported for the inhibition of human tissue kallikrein (hK1) by aprotinin with Ki and Kii values of 16.20 nM and 1.10 nM, respectively. It is noteworthy that as the Kii < Ki, the second aprotinin molecule binds to the enzyme with a larger affinity suggesting that the second binding site was probably created or positively modulated as a consequence of the binding of the first aprotinin molecule. These results suggest different kinetic properties for the two tissue kallikreins. The second binding site for aprotinin in rK1 and also in hK1 may have important implications for the physiological activity of these enzymes. This study also presents a review of the literature on parabolic competitive inhibition.
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