STRUCTURAL ENVIRONMENT OF AN ESSENTIAL CYSTEINE RESIDUE OF XYLANASE FROM CHAINIA SP (NCL-82.5.1)

N-(2,4-Dinitroanilino)maleimide (DAM) reacts covalently with the thiol group of the xylanase from Chainia leading to complete inactivation i n a manner similar to N-ethylmaleimide, but provides a reporter group at the active site of the enzyme. Increasing amounts of xylan offered enhanced protection against inactivation of the xylanase by DAM. Xylan (5 mg) showed complete protection, providing evidence for the presenc e of cysteine at the substrate-binding site of the enzyme. Kinetics of chemical modification of the xylanase by DAM indicated the involvemen t of 1 mol of cysteine residue per mol of enzyme, as reported earlier [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177] . The second-order rate constant for the reaction of DAM with the enzy me was 3.61 x 10(3) M(-1) min(-1). The purified xylanase was alkylated with DAM and digested with pepsin. The peptides were separated by gel filtration. The specific modified cysteinyl peptide was further purif ied by reverse-phase HPLC. The active-site peptide was located visuall y by its predominant yellow colour and characterized by a higher A(340 ) to A(210) ratio. The modified active-site peptide has the sequence: Glu-Thr-Phe-Xaa-Asp. The sequence of the peptide was distinctly differ ent from that of cysteinyl peptide derived from a xylanase from a ther motolerant Streptomyces species, but showed the presence of a conserve d aspartic acid residue consistent with the catalytic regions of other glucanases.