产碱杆菌Alcaligenes sp.KS-85来源肌酸酶活性中心的关键氨基酸功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0830

摘要/Abstract

摘要：

肌酸酶（Creatinase,EC 3.5.3.3）水解肌酸生成尿素和肌氨酸,是肌酐多酶级联检测中的关键酶。为进一步解析产碱杆菌来源肌酸酶的催化机理,利用蛋白质同源建模、分子对接、丙氨酸扫描技术分析了酶与底物的相互作用,并聚焦于酶活性中心4个功能未知的保守位点Phe64、Asp102、Phe252、Phe321,通过将氨基酸残基定点突变为6种具有代表性的氨基酸,结合生化实验对其功能进行解析。经研究,所有突变体的k_cat值大幅度降低;除6个突变体：F252A、F252S、F252Y、F252W、F321A、F321Y的K_M值降低,其余突变体的K_M均提高。结构分析表明Phe252通过与Tyr259形成的π-π堆积稳定酶-底物复合体;Asp102通过与Arg66,Gly322的氢键相互作用稳定酶反应的过渡态;Phe321,Phe64位于底物两侧,通过疏水侧链的排斥作用及空间位阻影响底物的定位。本研究通过对活性中心氨基酸残基的功能解析,为肌酸酶的催化机制解析和分子改造奠定了基础。

关键词: 肌酸酶, 活性中心, 催化效率, 分子对接, 功能氨基酸

Abstract:

Creatinase（CRE）hydrolyses creatine to urea and sarcosine,and plays an important role in the enzymatic measurement of creatinine concentration for in vitro diagnosis. To better understand the catalytic mechanism of CRE,the homology modeling,molecular docking and alanine scanning were used to analyze the interaction between enzyme and substrate. Focusing on 4 function-unknown key residues Phe64,Asp102,Phe252,and Phe321 in the active center of the enzyme,site-directed mutagenesis was conducted to mutate them into 6 representative amino acids,and their functions were analyzed by combing the bio-chemical experiment. The k_cat values of all mutants significantly decreased compared with the wild type enzyme. The K_M of F252A,F252S,F252Y,F252W,F321A,and F321Y decreased while that of others increased. Structural analysis showed that Phe252 stabilized the enzyme-substrate complex by the π-π stacking with Tyr259. Asp102 stabilized the transition state of the enzyme reaction by interacting with the hydrogen bonds of Arg66 and Gly322. Phe321 and Phe64 were located at two sides of the substrate,which affected substrate positioning by their hydrophobicity and steric hindrance. This study lays a foundation for understanding the catalytic mechanism and molecular engineering of creatinase via the functional analysis of amino acid residues in the active center.

Key words: creatinase, active center, catalytic efficiency, molecular docking, functional residues

郝俊尧, 马富强, 杨广宇. 产碱杆菌Alcaligenes sp.KS-85来源肌酸酶活性中心的关键氨基酸功能研究[J]. 生物技术通报, 2021, 37(3): 75-83.

HAO Jun-yao, MA Fu-qiang, YANG Guang-yu. Functional Analysis of Key Residues in the Active Center of Creatinase from Alcaligenes sp. KS-85[J]. Biotechnology Bulletin, 2021, 37(3): 75-83.

图/表 8

图1 肌酸酶反应机理图（1）His232从水分子中提取质子;（2）提取质子产生的OH-与底物的C1结合;（3）底物复合体夺取His232的质子;（4）夺取质子后形成的胍基水合物四面体结构不稳定,崩溃后生成肌氨酸和尿素

图2 建模CRE三维结构的活性中心 A：建模CRE的三维结构;B：Al-CRE三维结构（茶色）与底物直接作用的氨基酸残基,A,B代表不同单体;C：Al-CRE三维结构（茶色）活性中心的其他氨基酸残基

图3 酶活性口袋氨基酸残基与底物的作用力底物肌酸及相互作用的氨基酸分子以球棍模型显示,虚线表示氢键相互作用,虚线上的数字表示原子之间的距离,字母A,B表示不同的单体来源,未显示结构的氨基酸为疏水的非配体残基

图4 活性中心氨基酸位点丙氨酸扫描

表1 Al-CRE活性中心已知功能的氨基酸残基

氨基酸残基	位置	功能
Arg66	loop	底物结合
Ile84	3₁₀ helix	稳定过渡态
His233	loop	酸碱催化
Tyr259	β sheet	稳定过渡态
Glu263	β sheet	底物结合
His325	loop	稳定过渡态
Arg336	loop	底物结合
Glu359	β sheet	稳定过渡态
His377	β sheet	稳定过渡态

图5 Phe64、Asp102、Phe252、Phe321突变体SDS-PAGE电泳验证 M：蛋白分子量标准;A：Phe64突变体（1-6：F64A、F64S、F64D、F64R、F64Y、F64W）;B：Asp102突变体（1-6：D102A、D102S、D102R、D102Y、D102F、D102W）;C：Phe252突变体（1-6：F252A、F252S、F252D、F252R、F252Y、F252W）;D：Phe321突变体（1-6：F321A、F321S、F321D、F321R、F321Y、F321W）

表2 肌酸酶野生型及24个突变体动力学常数表

突变体	k_cat（min^-1）	K_M（mM）	k_cat/K_M（mM^-1·min^-1）
WT	108.7±1.43	2.12±0.33	51.27
F64A	13.603±2.06	125.00±3.02^a	0.11
F64S	30.66±1.66	282.20±19.61^a	0.11
F64D	ND	ND	ND
F64R	17.91±1.04	98.57±11.21^a	0.18
F64Y	28.47±1.38	10.15±1.85	2.80
F64W	37.84±0.68	13.82±0.97	2.73
D102A	8.28±0.52	29.1±2.69	0.28
D102S	1.056±0.07	51.64±3.56	0.05
D102R	1.60±0.12	27.37±8.26	0.05
D102F	4.97±0.04	37.85±14.77	0.13
D102Y	ND	ND	ND
D102W	13.13±0.10	25.78±0.94	0.50
F252A	1.60±0.037	1.36±0.099	1.18
F252S	2.50±0.033	0.46±0.03	5.47
F252D	ND	ND	ND
F252R	ND	ND	ND
F252Y	11.92±0.12	0.836±0.01	14.25
F252W	0.402±0.03	0.931±0.22	0.43
F321A	1.34±0.09	1.72±0.15	0.77
F321S	2.42±0.02	6.68±0.27	0.36
F321D	0.54±0.01	20.64±1.30	0.03
F321R	0.62±0.97	3.959±0.52	0.16
F321Y	2.15±0.02	0.51±0.05	4.25
F321W	0.32±0.01	23.35±2.09	0.01

图6 Phe64（A）、Asp102（B）、Phe252（C）、Phe321（D）氨基酸位点功能分析底物肌酸显示为棍棒结构,氨基酸显示为球棍模型,氢键相互作用表示为虚线

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