Mechanism of action of uridine diphosphoglucose dehydrogenase. Evidence for a second reversible dehydrogenation step involving an essential thiol group.
Although the enzyme UDP-glucose dehydrogenase from beef liver (E.C. 1.1.1.22) is known to abstract the pro-R hydrogen stereospecifically at carbon 6 of the glucose moiety of the substrate by a reversible step in converting UDP-glucose to UDP-alpha-D-gluco-hexodialdose (UDP-Glc-6-CHO), prolonged incubation of the enzyme with UDP-glucose and tritium-labeled NADH, under conditions favoring hydrogen exchange between the two, results in equivalent labeling of both hydrogens at carbon 6. This shows that the pro-S hydrogen at carbon 6 is also abstracted by a reversible process which must then involve a derivative of the carboxyl group of UDP-glucuronic acid (UDP-GlcUA) that is capable of reversible hydrogenation-dehydrogenation. It is the hydrolysis of this derivative that accounts for the well known irreversibility of the overall reaction. Derivatization of the enzyme's essential thiol group with 5,5'-dithiobis-(2-nitrobenzoate) eliminates the ability of the enzyme to either oxidize or reduce UDP-Glc-6-CHO. Replacement of the 5-thio-2-nitrobenzoate group with cyanide fully restores the enzyme's capacity to reduce UDP-Glc-6-CHO but has no effect on the inhibition of the oxidation to UDP-GlcUA. This indicates that the essential thiol group is involved in the second reversible dehydrogenation step and serves to form a thiol ester with the carboxyl of the product, UDP-GlcUA. It is suggested that thiol ester intermediates are a general characteristic of all 4-electron NAD-linked dehydrogenase reactions.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004228:Dithionitrobenzoic Acid; D005557:Formaldehyde; D008099:Liver / Q000201:enzymology; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis; D014316:Tritium; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D009243:NAD; D014316:Tritium; D005557:Formaldehyde; D004228:Dithionitrobenzoic Acid; D000429:Alcohol Oxidoreductases; D014533:Uridine Diphosphate Glucose Dehydrogenase