Temperature dependence of the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry.
The rates of the hydrolysis of N-acetylglycine, N-acetyl-L-methionine, and N-acetyl-L-phenylalanine by porcine acylase I in 0.1M phosphate buffer, which is inhibited by acetate product formation, were monitored calorimetrically at temperatures between 15.2 and 45.3 degrees C 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 heat released by the reaction as a function of time with regard to the integrated Michaelis-Menten equation yields apparent values for k(cat) 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 Km and k(cat) were determined over the temperature range from 15.2 to 45.3 degrees C. The intrinsic values of Km exhibited very little variation over this temperature range while the intrinsic values of k(cat) exhibited an increase over the same temperature range. The heats of reaction also exhibited an increase with temperature over this range with an average heat capacity change of -94 Jmol(-1)K(-1) for the three substrates.
Stödeman
Magnus
M
Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
Schwarz
Frederick P
FP
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D013552:Swine; D013696:Temperature
D010649:Phenylalanine; C006379:N-acetylmethionine; D008715:Methionine; D000581:Amidohydrolases; C019665:amidase; C044228:N-acetylphenylalanine; D005998:Glycine; C006368:aceturic acid