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



M Stodeman, Frederick P. Schwarz


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 kcat and apparent values for Km that depend on the product inhibition constant. The apparent values for Km 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 Km to its value at zero substrate concentration, intrinsic values of Kmwere 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 kcat 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.
Analytical Biochemistry


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


Stodeman, M. and Schwarz, F. (2003), Temperature Dependence of the Kinetics of the Acylase Hydrolysis Reaction by Differential Stopped Flow Microcalorimetry, Analytical Biochemistry (Accessed May 21, 2024)


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Created September 30, 2003, Updated October 12, 2021