CHEMICAL MODIFICATION OF XYLANASE FROM ALKALOTHERMOPHILIC BACILLUS SPECIES - EVIDENCE FOR ESSENTIAL CARBOXYL GROUP

The role of carboxyl group in the catalytic action of xylanase (M(r) 3 5000) from an alkalothermophilic Bacillus sp. was delineated through k inetic and chemical modification studies using Woodward's Reagent K. T he kinetics of inactivation indicated that one carboxyl residue was es sential 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 r esidues. In the presence of protecting ligand, modification of one car boxyl 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 mod ification of carboxyl group. The kinetic analysis of the modified enzy me showed similar K-m and lower k(cat) values than the native enzyme i ndicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylana se from alkalothermophilic Bacillus revealed the presence of tryptopha ns in the active site (Deshpande, V, Hinge, J. and Rao, M. (1990) Bioc him. Biophys. Acta 1041, 172-177). This finding and present studies de monstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from a lkalothermophilic Bacillus sp.