Chemical modification of xylanases: evidence for essential tryptophan and cysteine residues at the active site.
N-Bromosuccinimide (NBS) completely inactivated xylanases from Chainia and alkalophilic and thermophilic (AT) Bacillus with a concomittant decrease in absorption at 280 nm and with second-order rate constants of 10,500 and 5000 M-1.min-1, respectively at pH 6.0 and 25 degrees C. The kinetic analysis of inactivation indicated that one and three tryptophan residues were essential for the xylanase activity from Chainia and Bacillus, respectively. The xylanases were also inhibited by 2-hydroxy-5-nitrobenzyl bromide (HNBB). The modification of cysteine residues by p-hydroxymercurybenzoate (PHMB) and N-ethylmaleimide did not cause a loss in activity of the xylanase from Bacillus, whereas that from Chainia was completely inactivated. The kinetics of inactivation revealed the involvement of one cysteine residue for xylanase from Chainia with a second-order rate constant of 50,000 M-1.min-1. The PHMB-modified enzyme failed to show the presence of titrable -SH groups. Xylan afforded complete protection against inactivation by NBS, HNBB and PHMB, indicating the involvement of tryptophan and cysteine residues at the substrate-binding region of the enzyme.
Deshpande
V
V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Hinge
J
J
Rao
M
M
D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006358:Hot Temperature; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D013302:Streptomyces / Q000201:enzymology*; D014364:Tryptophan; D043325:Xylan Endo-1,3-beta-Xylosidase
D004791:Enzyme Inhibitors; D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D014364:Tryptophan; D004228:Dithionitrobenzoic Acid; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase; D003545:Cysteine; D001974:Bromosuccinimide