IS THE RAPID-PHASE OF ENZYME CATALYTIC CYCLES ENTROPY-DRIVEN?

测定了α -胰凝乳蛋白酶（CT）催化n -苯甲酰- l-酪氨酸乙酯（BYE）水解时的产热。所使用的仪器本质上是一个带有热电偶阵列作为反应检测器的止流装置，可以测量< 1 mK的温度变化，响应时间< 5 ms [Kodama， T.& Kometani， K.(1990) Thermochim]。《163105 - 110年)。仪器的死区时间为13 ms。热记录是在100 mM KCl中获得的，其中含有11% （v/v）乙腈作为助溶剂，并通过曲线拟合程序进行分析，该程序基于在可比条件下单独实验估计的稳态动力学参数（BYE与CT结合的平衡常数，CT与乙醇同时释放的酰化速率常数和CT的去酰化速率常数）[Berezin， I.V, Kazanskaya， N.F, & Klyosov， A.A.(1971) 15,121-124]。这些中间步骤的焓变作为可调参数，它们的最佳拟合值表明BYE与CT的结合是强吸热的，其次是强放热的CT酰化。限速的去酰化反应是吸热的，这使得整个反应几乎是热中性的。

Measurements were made of heat production accompanying hydrolysis of N-benzoyl-L-tyrosine ethyl ester (BYE) catalyzed by alpha-chymotrypsin (CT). The instrument used was essentially a stopped-flow device with a thermocouple-array as the reaction detector that can measure temperature changes < 1 mK with a response time < 5 ms [Kodama, T.& Kometani, K.(1990) Thermochim. Acta 163,105-110]. The dead time of the instrument was 13 ms. The heat records were obtained in 100 mM KCl containing 11% (v/v) acetonitril as cosolvent and analyzed by a curve-fitting procedure based on steady-state kinetic parameters estimated in separate experiments under comparable conditions (the equilibrium constant for BYE binding to CT,rate constants for acylation of CT with concomitant release of ethanol and deacylation of CT) [Berezin, I.V., Kazanskaya, N.F., & Klyosov, A.A.(1971) 15,121-124]. The enthalpy changes for these intermediate steps were taken as adjustable parmeters and their best fit values indicate that BYE binding to CT is strongly endothermic, followed by strongly exothermic CT acylation. The rate-limiting deacylation is endothermic, which makes the overall reaction almost thermally neutral.