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

酶催化循环的快速阶段是熵驱动的吗？

• 批准号: 06680656
• 负责人: KODAMA Takao
• 金额: $ 1.34万
• 依托单位: KYUSHU INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680656/
• 关键词: thermocouple-array; stopped-flow calorimeter; chymotrypsin; simulation; intermediate steps; acylation; deacylation; peptide substrate; 酵素反応サイクル; 熱力学的駆動力; エントロピー; エンタルピー

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.

测定了α-糜蛋白酶（CT）催化N-苯甲酰基-L-酪氨酸乙酯（BYE）水解过程中的产热。所使用的仪器基本上是具有热电偶阵列作为反应检测器的停流装置，其可以测量温度变化< 1 mK，响应时间< 5 ms [Kodama，T.&科梅塔尼湾（1990）Thermochim. Acta 163，105 -110]。该仪器的死时间为13 ms。在含有11%（v/v）乙腈作为共溶剂的100 mM KCl中获得热记录，并通过基于在可比条件下单独实验中估计的稳态动力学参数的曲线拟合程序进行分析（BYE与CT结合的平衡常数，伴随乙醇释放的CT酰化和CT脱酰化的速率常数）[Berezin，I. V.，卡赞斯卡亚，N.F.，& Klyosov，A.A.（1971）15，121 -124]。将这些中间步骤的焓变作为可调节参数，并且它们的最佳拟合值表明BYE与CT结合是强吸热的，然后是强放热的CT酰化。限速脱酰是吸热的，这使得整个反应几乎是热中性的。

项目成果

Y.Morita,M.Okamoto: "Development of Biosimulator for Analyzing Nonlinear Reaction Network" Proceeding of the 5th.Intl.Symp.of Process System Engng.1. 641-647 (1994)

Y.Morita，M.Okamoto：“用于分析非线性反应网络的生物模拟器的开发”第五届国际过程系统工程会议录1。

Harada, K., Yoshida, S., Ohtsubo, K., Komatsu, H., and Kodama, T.: "Stopped-flow calorimetry of an alpha-chymotrypsin reaction" Biochemistry. to be submitted.

Harada, K.、Yoshida, S.、Ohtsubo, K.、Komatsu, H. 和 Kodama, T.：“α-胰凝乳蛋白酶反应的停流量热法”生物化学。

H.Komatsu & K.Tawada: "Trinitrophenylation of the Reactive Lysine Residue in Double-Headed Myosin in the Presence of PPi" J.Biochem.115. 1190-1196 (1994)

小松H

M.Suzuki,J.Shigematsu & T.Kodama: "A hydration study of proteins in solution by Microwave dielectlic analysis" J.Phys.Chem.(in press).

铃木先生,重松先生

Harada,K.,Yoshida,S.,Ohtsubo,K.,Komatsu,H.,and Kodama,T.: "Stopped-flow calorimetry of an a-chymotrypsin reaction" Biochemistry. to be submitted.

Harada,K.、Yoshida,S.、Ohtsubo,K.、Komatsu,H. 和 Kodama,T.：“α-胰凝乳蛋白酶反应的停流量热法”生物化学。