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 in 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 presence of cysteine at the substrate-binding site of the enzyme. Kinetics of chemical modification of the xylanase by DAM indicated the involvement 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 enzyme 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 purified by reverse-phase HPLC. The active-site peptide was located visually by its predominant yellow colour and characterized by a higher A340 to A210 ratio. The modified active-site peptide has the sequence: Glu-Thr-Phe-Xaa-Asp. The sequence of the peptide was distinctly different from that of cysteinyl peptide derived from a xylanase from a thermotolerant Streptomyces species, but showed the presence of a conserved aspartic acid residue consistent with the catalytic regions of other glucanases.
Rao
M
M
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Khadilkar
S
S
Bandivadekar
K R
KR
Deshpande
V
V
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D008301:Maleimides; D010446:Peptide Fragments; C073040:N-(2,4-dinitroanilino)maleimide; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D003545:Cysteine