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 inhibitor (alpha-1 PI) was studied under second-order conditions by continuously 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 Pt = (kcat[S]/kiKm)In[ki[E]0t + 1], where ki is the second-order rate constant for the reaction, E + alpha-1 PI-->E* alpha-1 PI (inactive). ki 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 extension of the second-order progress curve approach to cover unequimolar mixtures of E and I is also offered.
Ozer
I
I
Department of Biochemistry, School of Pharmacy, Hacettepe University, Ankara, Turkey.
D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013223:Statistics as Topic; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D005456:Fluorescent Dyes; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D014357:Trypsin