Flow microcalorimetric study of enzyme reactions - Application to arylesterase from human serum

被引:6
作者
Debord, J
Harel, M
Bollinger, JC
Verneuil, B
Merle, L
Dantoine, T
机构
[1] Hop Dupuytren, Serv Pharmacol Toxicol, F-87042 Limoges, France
[2] Fac Med Limoges, Pharmacol Lab, F-87025 Limoges, France
[3] Inst Univ Format Maitres, F-87036 Limoges, France
[4] CNRS, UMRC, F-55830 Toulouse, France
[5] Fac Sci, Lab Sci Eau & Environm, F-87060 Limoges, France
[6] Fac Sci, Lab Chim Substances Nat, F-87060 Limoges, France
[7] Hop Dupuytren, Serv Gerontol Clin, F-87042 Limoges, France
关键词
arylesterase; paraoxonase; microcalorimetry; integrated Michaelis equation;
D O I
10.1016/j.tca.2004.08.018
中图分类号
O414.1 [热力学];
学科分类号
摘要
The enzymatic hydrolysis of phenyl acetate, catalysed by arylesterase/paraoxonase (EC 3.1.8.1) was studied at 37degreesC in Tris buffer, pH 8, by spectrophotometry and flow microcalorimetry, using an enzyme purified from human serum. After correction for buffer protonation and product ionization, the hydrolysis reaction was found to be slightly endothermic, with DeltaH = 8.2 kJ mol(-1). Microcalorimetric data were analysed with the integrated Michaelis equation to give the kinetic parameters of the enzyme: Michaelis constant K-m = 2.4 mM, catalytic constant k(cat) = 2.4 x 10(3) s(-1), bimolecular rate constant k(s) = 1.0 x 10(6) M-1 s(-1). These results were in agreement with the spectrophotometric method. This study confirms the usefulness of microcalorimetry in the field of enzyme kinetics. (C) 2004 Elsevier B.V. All rights reserved.
引用
收藏
页码:85 / 91
页数:7
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