Kinetic mechanism for stimulation by monovalent cations of the amidase activity of the plasma protease bovine activated protein C.
A study of the effect of monovalent cations on the steady-state kinetic parameters for the hydrolysis of the synthetic substrate N alpha-benzoyl-L-arginine-p-nitroanilide by activated bovine plasma protein C (APC) has been undertaken. The enzyme displayed a strict requirement for monovalent cations in its expression of amidolytic activity toward this substrate. Analysis of the variation in initial hydrolytic reaction rates, as a function of metal ion concentrations, suggested that at least two cation sites, or classes of sites, were necessary for catalysis to occur. After examination of the rate equations consequential to many different enzymic mechanisms that could account for these kinetic data, a mechanism was developed that fit the great majority of the experimental observations. In this mechanism it is postulated that cations bind to the enzyme in pairs, with a kinetically observable single binding constant, either preceded by or followed by binding of substrate. Catalysis occurs only after the enzyme-(metal cation)2-substrate complex is assembled. Some physical support for this mechanism was obtained upon the discovery that the binding (dissociation) constant for a competitive inhibitor of APC, p-aminobenzamidine, as determined by kinetic methodology, was independent of the concentration of Na+ and Cs+.
Steiner
S A
SA
Castellino
F J
FJ
D000581:Amidohydrolases / Q000097:blood*; D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002414:Cations, Monovalent; D002417:Cattle; D002586:Cesium / Q000494:pharmacology; D010450:Endopeptidases / Q000097:blood*; D004789:Enzyme Activation; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D011486:Protein C; D012697:Serine Endopeptidases; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D001798:Blood Proteins; D002414:Cations, Monovalent; D006023:Glycoproteins; D011486:Protein C; D002586:Cesium; D012965:Sodium Chloride; D001586:Benzoylarginine Nitroanilide; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000581:Amidohydrolases; D002118:Calcium