%0 Journal Article %A Webb, Steven R. %A Lee, Hung %T Characterization of xylose reductase from the yeast Pichia stipitis: Evidence for functional thiol and histidyl groups %D 1992 %J Microbiology, %V 138 %N 9 %P 1857-1863 %@ 1465-2080 %R https://doi.org/10.1099/00221287-138-9-1857 %I Microbiology Society, %X Xylose reductase (EC 1.1.1.21) from the yeast Pichia stipitis 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 pK app 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 p-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 mM-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 P. stipitis xylose reductase. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-138-9-1857