Importance of Product Inhibition in the Kinetics of the Acylase Hydrolysis Reaction by Differential Stopped Flow Microcalorimetry

Published

September 15, 2002

Author(s)

M Stodeman, Frederick P. Schwarz

Abstract

The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35 K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K_m) and the catalytic constant (K_cat) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k_cat were up to 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different initial substrate concentration reaction mixtures. In addition, values for K_{m were observed to increase linearly with increase in the initial substrate concentration. When an acetate product inhibition constant of 600 31 M}u-1^ was determined by isothermal titration calorimetry was used in the progress curve analysis, values for K_m and k_cat were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, δ_rH (cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from 4.69 0.009 kJ mol¹ for N-acetyl-L-phenylalanine substrate to -1.87 0.23 kJ mol^-1 for the N-acetyl-L-methionine.

Citation

Analytical Biochemistry

Volume

308

Issue

2

Pub Type

Journals

Keywords

enzyme kinetics, N-acety1L-amino acids, porcine kidney acylase I, reaction enthalpies, stopped flow microcalorimetry

Citation

Stodeman, M. and Schwarz, F. (2002), Importance of Product Inhibition in the Kinetics of the Acylase Hydrolysis Reaction by Differential Stopped Flow Microcalorimetry, Analytical Biochemistry (Accessed September 10, 2024)