1887

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Volume 138, Issue 9

Characterization of xylose reductase from the yeast : Evidence for functional thiol and histidyl groups Free

Abstract

Xylose reductase (EC 1.1.1.21) from the yeast NRC 2548 was purified to homogeneity via a two-step protocol using anion-exchange and gel-filtration chromatography. The pH-activity profile revealed the presence of two ionizable groups with p values of 5.8 and 8.1, suggesting the catalytic involvement of histidyl and thiol groups, respectively. Additional evidence supporting the involvement of these residues was provided by the use of group-specific inhibitors. The enzyme was rapidly inactivated in a pseudo-first order manner by the thiol-specific modifier -chloromercuriphenylsulphonate (PMBS) and analysis of the order-of-reaction suggested that one essential cysteine residue was modified to effect inactivation. Treatment of the enzyme with another thiol-specific modifier, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), showed that modification of one cysteine per monomer led to 90% loss of activity, further supporting the existence of one essential cysteine residue. Inactivation by PMBS was reversed by adding 1 m-β-mercaptoethanol. Inactivation of xylose reductase by the histidine-specific modifier diethylpyrocarbonate (DEP) followed a pseudo-first order process, and analysis of the order-of-reaction suggested that one essential histidine residue was modified to effect inactivation. Treatment of DEP-inactivated enzyme with 0.2 M-neutral hydroxylamine resulted in the recovery of 45% of enzyme activity. Protection of xylose reductase from PMBSand DEP-inactivation was provided by NADPH and NADH but not by NADP, D-xylose or DL-glyceraldehyde. This suggests that the essential histidine and cysteine residues may be involved with binding of cofactor by the xylose reductase.

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© Society for General Microbiology, 1992
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1992-09-01
2024-04-25
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Characterization of xylose reductase from the yeast Pichia stipitis: Evidence for functional thiol and histidyl groups
Microbiology 138, 1857 (1992); https://doi.org/10.1099/00221287-138-9-1857
/content/journal/micro/10.1099/00221287-138-9-1857
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