等温滴定量热法测定酶催化反应的热动力学参数

摘要

采用等温滴定量热法(ITC)测定猪肺血管紧张素转化酶（angiotensin converting enzyme，ACE）催化水解其体外模拟底物马尿酰-组氨酰-亮氨酸（Hip-His-Leu，HHL）反应的热动力学参数，考察了温度对动力学参数的影响。结果表明，该反应的摩尔水解焓ΔHhydr为正值，是吸热反应，且随温度升高ΔHhydr增大，等压比热容 cp为0.2126kJ/(mol·K)；ACE 催化 HHL 的水解反应符合 Michaelis-Menten 机理，在实验温度范围内（298.15～313.15K），米氏常数Km随温度升高而减小，催化常数kcat随温度的升高先增大后减少，在308.15K时达到最大值2.534s−1。将该法与传统的初始速率法进行比较，传统法存在的局限性使测得的 Km相对偏大。同时使用 ITC结合动力学分析测得ACE抑制剂药物依那普利拉为竞争性抑制剂，抑制常数KI为12.1 nmol/L，与文献比较证明该法可用于抑制剂类型的判断，是一种开发 ACE 抑制剂的新方法。应用该方法确定活性多肽Arg-Tyr-Leu-Gly-Tyr（RY-5)为非竞争性抑制剂，抑制常数KI为1.0μmol/L。%Thermodynamic and kinetic parameters of angiotensin converting enzyme(ACE)catalyzed hydrolysis of simulating substrate Hippuryl-Histidyl-Leucine(HHL)in vitro were determined by isothermal titration calorimetry(ITC). The effect of temperature on kinetic parameters was investigated; the results showed that the ACE-catalyzed reaction was endothermic with a small constant pressure specific heat capacity [cp=0.2126kJ/(mol·K)]. The value of molar hydrolysis enthalpyΔHhydrwas positive and increased as temperature rose. The reaction mechanism was in accordance with the Michaelis-Menten model in the temperature range(298.15—313.15K);the effect of temperature on the Michaelis constant(Km)was negative,while catalytic constant(kcat)first increased then decreased with the increase of temperature,reaching the maximum value of 2.534s−1 at 308.15K. Initial rate method was also used in order to compare with ITC method. TheKmmeasured by the initial rate method was relatively large because of the limitation in itself. The inhibitory type of the drug enalapril, known as ACE inhibitor,was determined by ITC and enzymatic kinetics analysis. The results show thatthe inhibitor was a competitive inhibitor with inhibition constantKI=12.1nmol/L. The ITC method is applicable for determining the inhibitor type in comparison with literature. It is a new approach for the development of ACE inhibitors. We determined that the active peptides Arg-Tyr-Leu-Gly-Tyr (RY-5) were noncompetitive inhibitors withKIof 1.0μmol/L by using this method..