Kinetic analysis of enzyme inactivation under second-order conditions by use of substrate-to-product progress curves: Application to the inhibition of trypsin by alpha-1 proteinase inhibitor

The inhibition of bovine pancreatic trypsin by human alpha-1 proteinase inh ibitor (alpha-1 PI) was studied under second-order conditions by continuous ly monitoring the fluorescence change due to the enzymatic hydrolysis of N alpha-benzoyl-L-arginine 7-amido-4-methyl-coumarin as substrate. Employing equimolar starting concentrations of enzyme and inhibitor (110-220 nM), the fluorescence progress curve was analyzed according to the equation P-t = ( k(cat)[S]/k(i)K(m))ln{k(i)[E](0)t + 1}, where hi is the second-order rate c onstant for the reaction, E + alpha-1 PI --> E*alpha-1 PI (inactive). k(i) was found to be 1.8 +/- 0.16 x 10(7) M-1 min(-1) (at pH 7.0 and 25 degrees C), in close agreement with results obtained by alternative kinetic methods . The method reported appears to be valid and should be useful in the study of fast reactions where one of the reaction partners is an enzyme. An exte nsion of the second-order progress curve approach to cover unequimolar mixt ures of E and I is also offered. (C) 1998 Academic Press.