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

Abstract 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 P 2 . The different points obtained at different nucleophile concentrations are situated on a straight line with intercepts K s on the K m axis and k 3 [E] 0 on the V axis. Since the Eisenthal and Cornish-Bowden method is considered as the best graphic method for determination of the kinetic parameters K m and V of enzyme reactions, the graphic procedure proposed here for determination of k 2 , k 3 , k 4 and K s by the method of added nucleophile is to be preferred. This procedure was applied for determination of the individual constants of the hydrolysis of N -acetylated l -amino acid methyl esters catalysedby alkaline mesentericopeptidase.