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

Published

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 (Km) and the catalytic constant (Kcat) 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 kcat 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 Km were observed to increase linearly with increase in the initial substrate concentration. When an acetate product inhibition constant of 600 31 Mu-1^ was determined by isothermal titration calorimetry was used in the progress curve analysis, values for Km and kcat were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, δrH (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

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 May 18, 2024)

Issues

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Created September 14, 2002, Updated October 12, 2021