Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane.
In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.
Turner
William
W
Institute for Plant Biotechnology, University of Stellenbosch, Matieland, South Africa.
Botha
Frederik C
FC
D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D031786:Saccharum / Q000737:chemistry*; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014535:Uridine Diphosphate Glucuronic Acid / Q000494:pharmacology; D014540:Uridine Diphosphate Xylose / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009243:NAD; D014535:Uridine Diphosphate Glucuronic Acid; D014540:Uridine Diphosphate Xylose; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014532:Uridine Diphosphate Glucose