Active site characterization of the exo-N-acetyl-β-D-glucosaminidase from thermotolerant Bacillus sp NCIM 5120:: involvement of tryptophan, histidine and carboxylate residues in catalytic activity

被引：10

作者：

Amutha, B ^{[1
]}

Khire, JM ^{[1
]}

Khan, MI ^{[1
]}

机构：

[1] Natl Chem Lab, Div Biochem Sci, Pune 411008, Maharashtra, India

来源：

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS | 1999年 / 1427卷 / 01期

关键词：

N-acetylglucosaminidase; active site tryptophan residue; active site histidine residue; active site carboxylate residue;

D O I：

10.1016/S0304-4165(99)00009-4

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The exo-N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) from thermotolerant Bacillus sp. NCIM 5120 is a homotetramer with a molecular mass of 240 000 kDa. Chemical modification studies on the purified exo-N-acetyl-beta-D-glucosaminidase revealed the involvement of a single tryptophan, histidine and carboxylate, per monomer, in the catalytic activity of the enzyme. Spectral analysis and maintenance of total enzyme activities indicated that N-acetylglucosamine (competitive inhibitor) and p-nitrophenyl-N-acetyl-beta-D-glucosaminide (substrate) prevented the modification of a single essential tryptophan, histidine and carboxylate residue. Kinetic parameters of partially inactivated enzyme (by NBS/HNBB) showed the involvement of tryptophan in substrate binding while that of histidine (by photooxidation/DEPC) and carboxylate (by EDAC/WRK) in catalysis. The Bacillus sp. NCIM 5120 exo-N-acetyl-beta-D-glucosaminidase deviates from the reported N-acetyl-beta-D-glucosaminidases and beta-hexosaminidases that utilize anchimeric assistance in their hydrolytic mechanism. (C) 1999 Elsevier Science B.V. All rights reserved.

引用

页码：121 / 132

页数：12

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