热动力学的滴定量热法研究 Ⅱ.单底物酶促反应的热动力学  

Titration Calorimetry Applied to the Study of Thermokinetics II . Thermokinetics of Single - substrate Enzyme - catalyzed Reactions

作　　者：梁毅[1] 汪存信[2] 刘欲文[2] 屈松生[2] 邹国林[3] 

机构地区：[1]中国科学院生物物理研究所 [2]武汉大学化学与环境科学学院,武汉430072 [3]武汉大学生命科学学院,武汉430072

出　　处：《化学学报》2000年第3期308-312,共5页Acta Chimica Sinica

基　　金：国家自然科学基金(39970164;29873036;29773033);中国博士后科学基金;中国科学院王宽诚博士后工作奖励基金资助项目

摘　　要：用滴定量热法分别建立了滴定期和停滴反应期单底物酶促反应热动力学的数学模型.根据这两种模型,可由一次实验的滴定量热曲线同时解析出单底物酶促反应的热力学参数(△_rH_m)和动力学参数(K_m和k_2).用滴定量热法研究了一个经典的单底物酶促反应——过氧化氢酶催化分解过氧化氢反应的热动力学,由滴定期和停滴反应期热动力学模型解析出在298.15K和pH=7.0时该反应的米氏常数K_m分别为(5.41±0.24)×10^(-3)和(5.43±0.21)×10^(-3)mol·L^(-1),酶转换数k_2分别为(3.58±0.33)×10~3和(3.60±0.41)×10~3s^(-1),摩尔反应焓为(-86.75±0.88)kJ·mol^(-1),实验结果验证了上述热动力学模型的正确性.Titration calorimetry is emerging as an important tool for characterizing interactions of biological macromolecules by virtue of its general applicability, high accuracy and precision. In this paper, two mathematical models for thermokinetics of a single-substrate enzyme-catalyzed reaction in titration period and in the stopped-titration reaction period, respectively, have been developed, by using titration calorimetry. On the basis of the titration calorimetric curve, one can use these two models to calculate not only the thermodynamic data ( △rHm) but also the kinetic data (Km and k2) for the reaction. Thermokinetics of a well-studied single-substrate enzymatic reaction, the catalase-catalyzed decomposition of hydrogen peroxide, was thus investigated by titration calorimetry, and the molar enthalpy ( △rHm) was found to be ( - 86.75 × 0.88)kJ mol-1. The Michaelis constant (Km) for H2O2 and the turn - over number of the enzyme (k2) were determined by the titration-period thermokinetic model to be (5.41 ± 0.24) × 10-3 mol·L-1 and (3.58 ± 0.33) × 103s-1, respectively, whereas the corresponding kinetic parameters calculated by the stopped-titration-reaction-period thermokinetic model were (5.43 ±0.21) × 10-3 mol·L-1 and (3.60±0.41) × 103 s-1, respectively, at 298.15 K and pH 7.0. Reliability of the above thermokinetic models was verified by the experimental data.

关 键 词：滴定量热法 酶促反应 热动力学 过氧化氢酶 

分 类 号：O642[理学—物理化学]