Temperature Dependence of the Kinetics of the Acylase Hydrolysis Reaction by Differential Stopped Flow Microcalorimetry

Published

October 1, 2003

Author(s)

M Stodeman, Frederick P. Schwarz

Abstract

The rate of the hydrolysis of N-acetyl-L-methionine by porcine acylase I in 0.1 M phosphate buffer, which is reduced by acetate product inhibition, was monitored calorimetrically at 288.4 K, 298.4 K, 308.5 K, and 311.5 K by a differential stopped flow microcalorimeter. Since the acylase is thermally stable and the pH of the phosphate buffer changes by less than 0.01 pH units over this temperature range, any temperature effect on the hydrolysis reaction can be attributed to the effect of temperature on the kinetics of the reaction. Analysis of the integrated power released by the reaction as a function of time in terms of the integrated Michaelis-Menten equation yields apparent values for k_cat and apparent values for K_m that depend on the product inhibition constant. The apparent values for K_m also exhibited a dependence on the initial substrate concentration because of the acetate product inhibition at each temperature. By assuming that the inhibition constant is independent of temperature over this temperature range and from extrapolation of K_m to its value at zero substrate concentration, intrinsic values of K_mwere determined that ranged from 0.95 0.09 mM at 288.4 K to 1.23 0.40 mM at 308.5 K and intrinsic values of k_cat were determined that exhibited an increase from 11.7 1.7 s^-1 at 288.4 K to 18.9 6.5 S^-1 at 318.5 K. The exothermicity of the heat of reaction also increases from -1.43 0.12 kJ mol^-1 at 288.4 K to 3.75 0.24 kJ mol^-1 at 315.5 K.

Citation

Analytical Biochemistry

Volume

321

Issue

1

Pub Type

Journals

Keywords

differential stopped flow microcalorimet, enzyme kinetics porcine kidney acylase, N-acetyl-L-methionine, reaction enthalpies, temperature

Citation

Stodeman, M. and Schwarz, F. (2003), Temperature Dependence of the Kinetics of the Acylase Hydrolysis Reaction by Differential Stopped Flow Microcalorimetry, Analytical Biochemistry (Accessed December 15, 2024)