Transition-State Inhibition of Thrombin and Trypsin by Amidinophenylpyruvates

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Abstract
The interactions of human thrombin and bovine trypsin with 4-amidinophenylpyruvate (p-APPA), 3-amidinophenylpyruvate (m-APPA) and benzamidine were studied by equilibrium binding and by stopped-flow kinetics with proflavin displacement. The excellent inhibitory properties of p-APPA with both enzymes are explained by a two-step transition-state mechanism in which the initial Michaelis complex E .cntdot. I reacts rapidly to form a fairly stable, chemically bonded complex E-I, in which the keto group of p-APPA forms a hemiketal with the .gamma.-O-atom of Ser195 at the active site. The hemiketal complex of thrombin and p-APPA may be further stabilized by hydrogen bond between a carboxylate oxygen of p-APPA and the N.tau.-atom (= N.EPSILON.2) of His57, as was previously shown for the trypsin-p-APPA complex by X-ray crystal structure analysis by J. Walter and W. Bode. m-APPA is apparently sterically incapable of forming a hemiketal with Ser195O.gamma.; it does not bind to thrombin or trypsin in a time-dependent manner, and it displays KI values with both enzymes close to those obtained for benzamidine itself. In the p-APPA-thrombin reaction the overall binding constant KI (E-I) is 1.3 .mu.M, while the initial binding displays a KM (E .cntdot. I) estimated at least 100-fold higher (700 .mu.M). The half-time for the formation of E-I is about 0.6 s at a p-APPA concentration of 1 .mu.M.

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