Affinity alkylation of the Trp-b4 residue of the beta-subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp 130

	Affinity alkylation of the Trp-b4 residue of the beta-subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp 130
	Huang, X; Zeng, R; Ding, XM; Mao, X; Ding, Y; Rao, ZH; Xie, Y; Jiang, WH; Zhao, GP
刊名	JOURNAL OF BIOLOGICAL CHEMISTRY
	2002
卷号	277 期号:12 页码:10256-10264
通讯作者	Jiang, WH (reprint author), Chinese Acad Sci, Lab Microbial Mol Physiol, Inst Plant Physiol & Ecol, 345 Lingling Lu, Shanghai 200032, Peoples R China.,
英文摘要	Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the 6-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the,6-subunit, Trp-B4, was alkylated by BA-7-ACA. By H-1-C-13 HSQC spectroscopy of C130 labeled by BA-2-C-13-7-ACA it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K-m, decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.
学科主题	Biochemistry & Molecular Biology
类目[WOS]	Biochemistry & Molecular Biology
关键词[WOS]	PROTEINS ; PURIFICATION
收录类别	SCI
语种	英语
WOS记录号	WOS:000174549200087
内容类型	期刊论文
版本	出版稿
源URL	[http://202.127.25.143/handle/331003/2539]
专题	上海生化细胞研究所_上海生科院生化细胞研究所
推荐引用方式 GB/T 7714	Huang, X,Zeng, R,Ding, XM,et al. Affinity alkylation of the Trp-b4 residue of the beta-subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp 130[J]. JOURNAL OF BIOLOGICAL CHEMISTRY,2002,277(12):10256-10264.
APA	Huang, X.,Zeng, R.,Ding, XM.,Mao, X.,Ding, Y.,...&Zhao, GP.(2002).Affinity alkylation of the Trp-b4 residue of the beta-subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp 130.JOURNAL OF BIOLOGICAL CHEMISTRY,277(12),10256-10264.
MLA	Huang, X,et al."Affinity alkylation of the Trp-b4 residue of the beta-subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp 130".JOURNAL OF BIOLOGICAL CHEMISTRY 277.12(2002):10256-10264.