The role of carboxyl group in the catalytic action of xylanase (M(r) 35,000) from an alkalothermophilic Bacillus sp. was delineated through kinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M-1 min-1. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar Km and lower kcat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Deshpande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172-177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp.
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