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Biochim Biophys Acta. 1999 Mar 14;1427(1):121-32.

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

Author information

1
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India.

Abstract

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 240000 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.

PMID:
10082993
DOI:
10.1016/s0304-4165(99)00009-4
[Indexed for MEDLINE]

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