Determination of the individual rate and association constants of the hydrolysis catalysed by serine proteinases by means of added nucleophiles in Eisenthal and Cornish-Bowden coordinates.
It is shown that in the three-step enzyme-catalysed hydrolysis the addition of nucleophile shifts the common intersection point in Eisenthal and Cornish-Bowden coordinates when registering the second product P2. The different points obtained at different nucleophile concentrations are situated on a straight line with intercepts Ks on the Km axis and k3 [E]o on the V axis. Since the Eisenthal and Cornish-Bowden method is considered as the best graphic method for determination of the kinetic parameters Km and V of enzyme reactions, the graphic procedure proposed here for determination of k2, k3, k4 and Ks by the method of added nucleophile to be preferred. This procedure was applied for determination of individual constants on the hydrolysis of N-acetylated L-amino acid methyl esters catalysed by alkaline mesentericopeptidase.
D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D012697:Serine Endopeptidases
D010450:Endopeptidases; D012697:Serine Endopeptidases; C021553:mesentericopeptidase