Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity.
The endo-beta-1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.
Wen
L
L
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
Miao
Z W
ZW
Qing
W D
WD
D000596:Amino Acids / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry; D006902:Hydroxymercuribenzoates / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D013774:Tetranitromethane / Q000737:chemistry; D014250:Trichosporon / Q000201:enzymology*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D002264:Carboxylic Acids; D006902:Hydroxymercuribenzoates; D007093:Imidazoles; C030601:4-hydroxymercuribenzoate; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D013774:Tetranitromethane; C012160:N-acetylimidazole