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

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.