Identification of a glutamate residue at the active site of xylanase A from Schizophyllum commune.
The xylanase A (endo-1,4-beta-D-xylan xylanhydrolase) of the basidiomycete Schizophyllum commune was treated with the powerful carboxylate-modifying reagent 1-(4-azonia-4,4-dimethyl-pentyl)-3-ethylcarbodiimide iodide (EAC) in the presence of substrate. This treatment was followed by complete inactivation of the enzyme with [14c]EAC after the removal of excess reagent and protecting ligand. The inactivated enzyme was digested with endoproteinase Arg-C or trypsin, and peptides were separated and purified using reverse-phase high-performance liquid chromatography. Following sub-digestion of individual radioactive peptides with staphylococcal V8 protease and endoproteinase Lys-C, amino acid composition analysis and sequencing analysis revealed that the [14C]EAC label was bound exclusively to Glu87. Comparison of the primary sequences of related xylanase with that of xylanase A revealed that Glu87 is a highly conserved residue. Based on this similarity and the mechanism of carbodiimide action, Glu87 is proposed to act as the nucleophile in the catalytic mechanism of xylanase A. The possible environment of the putative catalytic glutamate residue was explored using hydrophobic-cluster analysis and secondary-structure prediction based on the primary sequence of xylanase.
Bray
M R
MR
Department of Microbiology, University of Guelph, Canada.
Clarke
A J
AJ
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D043364:Endo-1,4-beta Xylanases; D005971:Glutamates / Q000032:analysis / Q000378:metabolism*; D018698:Glutamic Acid; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012567:Schizophyllum / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013210:Staphylococcus / Q000201:enzymology
D002234:Carbodiimides; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; C044568:1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide; D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases