等温滴定量热法测定酶催化反应的热动力学参数

Thermodynamics and kinetics of an enzyme-catalyzed reaction determined by isothermal titration calorimetry

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ΔH_(hydr)为正值,是吸热反应,且随温度升高ΔH_(hydr)增大,等压比热容c_p为0.2126k J/(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K_m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K_m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K_I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K_I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=0.2126 kJ/（mol·K）]. The value of molar hydrolysis enthalpy ΔHhydr was 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 308.15 K. Initial rate method was also used in order to compare with ITC method. The Km measured 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 that the inhibitor was a competitive inhibitor with inhibition constant KI=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 with KI of 1.0μmol/L by using this method..