Functional Analysis of Key Residues in the Active Center of Creatinase from Alcaligenes sp. KS-85

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

Abstract

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

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.

Figures/Tables 8

References 23

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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

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

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

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