Essential carboxy groups in xylanase A.
An endo-1,4-beta-xylanase of Schizophyllum commune was purified to homogeneity through a modified procedure employing DEAE-Sepharose CL-6B and gel-filtration chromatography on Sephadex G-50. The role of carboxy groups in the catalytic mechanism was delineated through chemical modification studies. The water-soluble carbodi-imide 1-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodi-imide iodide (EAC) inactivated the xylanase rapidly and completely in a pseudo-first-order process. Other carbodi-imides and Woodward's Reagent K were less effective in decreasing enzymic activity. Significant protection of the enzyme against EAC inactivation was provided by a mixture of neutral xylo-oligomers. The pH-dependence of the EAC inactivation revealed the presence of a critical ionizable group with a pKa value of 6.6 in the active site of the xylanase. Treatment of the enzyme with diethyl pyrocarbonate resulted in modification of all three histidine residues in the enzyme with 100% retention of original enzymic activity. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetimide and p-chloromercuribenzoate indicated the absence of free/reactive thiol groups. Reaction of the xylanase with tetranitromethane did not result in a significant activity loss as a result of modification of tyrosine residues.
Bray
M R
MR
Department of Microbiology, University of Guelph, Ontario, Canada.
Clarke
A J
AJ
D001487:Basidiomycota / Q000201:enzymology*; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology; D002384:Catalysis; D003545:Cysteine; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D012567:Schizophyllum / Q000201:enzymology*; D014443:Tyrosine; D043325:Xylan Endo-1,3-beta-Xylosidase
D002234:Carbodiimides; D014443:Tyrosine; C044568:1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide; D006639:Histidine; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D003545:Cysteine