等温滴定量热法测定酶催化反应的热动力学参数

doi:10.16085/j.issn.1000-6613.2016.11.011

化工进展 ›› 2016, Vol. 35 ›› Issue (11): 3459-3464.DOI: 10.16085/j.issn.1000-6613.2016.11.011

等温滴定量热法测定酶催化反应的热动力学参数

彭尚, 孙丽霞, 熊珍爱, 周利琴, 兰雄雕, 孙建华, 童张法, 廖丹葵

广西大学化学化工学院, 广西高校资源化工应用新技术重点实验室, 广西南宁 530004

收稿日期:2016-04-27 修回日期:2016-07-04 出版日期:2016-11-05 发布日期:2016-11-05
通讯作者: 廖丹葵,教授,博士生导师。E-mail:liaodk@gxu.edu.cn
作者简介:彭尚(1988-),男,硕士研究生,从事化学工艺方面的研究。
基金资助:
国家自然科学基金面上项目（51372043）、广西教育厅科研项目（KY2016YB035）、广西大学博士启动项目（XBZ160130）及广西博士后专项资助项目。

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

PENG Shang, SUN Lixia, XIONG Zhen'ai, ZHOU Liqin, LAN Xiongdiao, SUN Jianhua, TONG Zhangfa, LIAO Dankui

School of Chemistry and Chemical Engineering, Guangxi University, Guangxi Colleges and Universities Key Laboratory of New Technology and Application in Resource Chemical Engineering, Nanning 530004, Guangxi, China

Received:2016-04-27 Revised:2016-07-04 Online:2016-11-05 Published:2016-11-05

摘要/Abstract

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0.2126kJ/（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K_m随温度升高而减小，催化常数k_cat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1.0 μmol/L。

关键词: 等温滴定量热, 反应动力学, 血管紧张素转化酶, 热力学, 生物催化

Abstract: 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 [c_p=0.2126kJ/(mol·K)]. The value of molar hydrolysis enthalpy ΔH_hydrwas 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 (K_m) was negative, while catalytic constant (k_cat) 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. The K_mmeasured 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 K_I=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 K_Iof 1.0μmol/L by using this method.

Key words: isothermal titration calorimetry, reaction kinetics, angiotensin converting enzyme, thermodynamic, biocatalysis

中图分类号:

O642.3

彭尚, 孙丽霞, 熊珍爱, 周利琴, 兰雄雕, 孙建华, 童张法, 廖丹葵. 等温滴定量热法测定酶催化反应的热动力学参数[J]. 化工进展, 2016, 35(11): 3459-3464.

PENG Shang, SUN Lixia, XIONG Zhen'ai, ZHOU Liqin, LAN Xiongdiao, SUN Jianhua, TONG Zhangfa, LIAO Dankui. Thermodynamics and kinetics of an enzyme-catalyzed reaction determined by isothermal titration calorimetry[J]. Chemical Industry and Engineering Progress, 2016, 35(11): 3459-3464.

参考文献

[1] ELLIS R J. Macromolecular crowding:obvious but underappreciated[J]. Trends Biochem. Sci.,2001,26(10):597-604.
[2] OLSEN S N. Application of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions[J]. Thermochim Acta,2006,448(1):12-18.
[3] LONHIENNE T G A,WINZOR D J. A potential role for isothermal calorimetry in studies of the effects of thermodynamic non-ideality in enzyme-catalyzed reactions[J]. Journal of Molecular Recognition,2004,17(5):351-361.
[4] CAI L F,CAO A N,LAI L H. An isothermal titration calorimetric method to determine the kinetic parameters of enzyme catalytic reaction by employing the product inhibition as probe[J]. Analytical Biochemistry,2001,299(1):19-23.
[5] BIANCONI M L. Calorimetry of enzyme-catalyzed reactions[J]. Biophysical Chemistry,2007,126(1/2/3):59-64.
[6] 宋熙熙. 等温滴定量热法测定脲酶催化尿素水解反应动力学[J]. 浙江大学学报,2009,36(2):175-179.
[7] 周敬豪,徐存华,李玉婵,等. 磁性壳聚糖微球的合成及其在固定化血管紧张素转化酶的应用[J]. 化工进展,2013,32(10):2440-2445.
[8] 周利琴,廖丹葵,孙建华. 猪肺血管紧张素转化酶的提取与纯化[J]. 南方农业学报,2014,45(4):639-642.
[9] TELLEZ-SANZ R,GARCIA-FUENTES L,BARON C. Calorimetric analysis of lisinopril binding to angiotensin Ⅰ—converting enzyme[J]. Febs Letters,1998,423(1):75-80.
[10] 刘宏,陈兰英. 血管紧张素转化酶纯化和性质研究[J]. 中国生物化学与分子生物学报,2000(6):788-792.
[11] 吴琼英,马海乐,崔恒林,等. 猪肺血管紧张素转化酶的提取纯化及其性质研究[J]. 食品科学,2004(9):71-73.
[12] ANDUJAR-SANCHEZ M,CAMAR-ARTIGAS A,JARA-PEREZ V. A calorimetric study of the binding of lisinopril,enalaprilat and captopril to angiotensin converting enzyme[J]. Biophysical Chemistry,2004,111(2):183-189.
[13] 彭益强,刘鹏,邓峰,等. 源于马铃薯的多酚氧化酶活性中心必需基团组成与抑制机理[J]. 化工进展,2012,31(2):406-411.
[14] CHENG Y,PRUSOFF W H. Relationship between the inhibition constant(Ki)and the concerntration of an inhibitor that causes a 50% inhibition (I50) of an enzymatic reaction[J]. Biochemical Pharmacology,1973,22(1):3099-3108.
[15] CECONI C,FRANCOLINI G,OLIVARES A,et al. Angiotensin converting enzyme (ACE) inhibitors have different selectivity for bradykinin binding sites of human somatic ACE[J]. European Journal of Pharmacology,2007,577(1/2/3):1-6.
[16] CONTRERAS M D,CARRON R,MONTERO M J,et al. Novel casein-derived peptides with antihypertensive activity[J]. International Dairy Journal,2009,19(10):566-573.

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

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