Loss of C‐5 hydrogen during oxidation of UDP‐D‐glucose by UDP‐D‐glucose dehydrogenase

Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction

“…Utilization of UDP- gluco -hexodialdose as substrate for oxidation and reduction, by contrast, could be reconciled with the concerted mechanism, because the observed activities may not reflect exactly the steps that occur in the normal reaction pathway. Using UDP- galacto -hexodialdose (prepared from UDP-galactose by oxidation with galactose oxidase) as surrogate of UDP- gluco -hexodialdose, the authors demonstrated that the C-5 tritium readily exchanges with the solvent proton in a spontaneous (non-enzymatic) reaction 19 . In contrast, chemically synthesized UDP- gluco -hexodialdose forms a hydrate upon contact with water, as shown by Campbell and Tanner 18 .…”

“…They also proposed that NADH was released from the enzyme complex with UDP- gluco -hexodialdose only after formation of the covalent thiohemiacetal adduct (Scheme 1). In their mechanistic studies of bovine liver UGDH, Feingold and colleagues observed that tritium label from C-5 of UDP-Glc was partly ‘washed out’ to solvent during the enzymatic reaction 19 . Loss of label (30% of the original tritium) was explained by an enolization of the C-6 aldehyde in UDP- gluco -hexodialdose prior to covalent adduct formation (Scheme 2).…”

“…We took an approach distinct from that of Feingold and co-workers 19 in that we measured incorporation of solvent deuterium by the UDP-GlcUA product. In situ proton NMR spectroscopy was employed to observe the enzymatic transformations in real time.…”

“…We performed in situ NMR experiments at pD 7.5 (wild type) and 8.3 (E161Q), because hUGDH is active and stable under these conditions. The previous work with bovine liver UGDH was done at a higher pH of 8.7 19 . We therefore analyzed deuterium incorporation into the UDP-GlcUA product at different pD values in the range 5.9–8.8, using NMR measurement at a single time point of the reaction.…”

“…The original observation made with bovine liver UGDH that tritium label at C-5 of UDP-Glc was partly washed out during reaction 19 was re-examined using an alternative, equally diagnostic approach in which deuterium uptake from solvent was measured. Instead of the bovine liver enzyme, the structurally and mechanistically well-characterized hUGDH, both in wild-type and Glu 161 →Gln mutated form, was used.…”