Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry.
The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K 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 a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), 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, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine.
Stödeman
Magnus
M
Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, Rockville, MD 20850, USA.
Schwarz
Frederick P
FP
D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D013816:Thermodynamics
C010530:N-acetylalanine; D010649:Phenylalanine; C006379:N-acetylmethionine; D008715:Methionine; D000581:Amidohydrolases; C019672:aminoacylase I; C044228:N-acetylphenylalanine; D000409:Alanine; D005998:Glycine; C006368:aceturic acid