NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.
An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.
Zepeda
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Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago.
Monasterio
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Ureta
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D000449:Aldehyde Reductase; D000818:Animals; D001667:Binding, Competitive; D002845:Chromatography; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification*; D013552:Swine
D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; C023142:polyol dehydrogenase (NADP)