Identification of an essential tyrosyl residue in the binding site of Schizophyllum commune xylanase A.
Ultraviolet difference spectroscopy studies with the Schizophyllum commune xylanase in the presence of inhibitors and substrates indicated the participation of one or more tyrosyl residues in the binding of substrates to xylanase. Chemical modification experiments with group-specific reagents in the absence and presence of substrates confirmed the essential role of a tyrosyl residue in substrate binding while discounting the participation of tryptophan. A fourth-derivative absorbance spectroscopic method was developed to facilitate the quantitation of modified tyrosyl and tryptophanyl residues. This analysis showed that two tyrosyl residues of the xylanase are modified by tetranitromethane in the absence of substrate with the concomitant loss of catalytic activity. Protection of the xylanase with xylooligosaccharides resulted in the nitration of only one residue, and such enzyme derivatives retained 94% catalytic activity. Differential modification of the xylanase with tetranitromethane generated an enzyme derivative with the characteristic absorbance at 428 nm of 3-nitrotyrosine. Amino acid analysis and N-terminal sequencing of peptides with strong absorbance at 428 nm isolated from the protease-digested modified enzyme by reverse-phase HPLC identified the essential residue as Tyr97. Alignment of the S. commune xylanase amino acid sequence with those of the 18 other known family G xylanases revealed that Tyr97 is a conserved aromatic residue, further suggesting its essential role in substrate binding.
Department of Microbiology, University of Guelph, Ontario, Canada.
M R
MR
Bray
A J
AJ
Clarke
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001974:Bromosuccinimide / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D043364:Endo-1,4-beta Xylanases; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012567:Schizophyllum / Q000201:enzymology*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000032:analysis* / Q000737:chemistry; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D014443:Tyrosine; D014364:Tryptophan; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases; D001974:Bromosuccinimide