Spectroscopic evidence for the formation of a 4-keto intermediate in the UDP-apiose/UDP-xylose synthase reaction.
Uridine diphospho-D-glucose (UDP-Glc) and UDP-methyl-D-glucuronate (UDP-GlcUAMe) have been shown to be competitive inhibitors for the UDP-apiose/udp-xylose synthase from cell suspension cultures of parsley. The apparent Ki values for these substrate analogues were of the same order of magnitude as the apparent Km value for the substrate UDP-D-glucuronic acid (UDP-GlcUA). The difference spectrum of the incubation mixture containing UDP-GlcUA, NAD+ and the highly purified enzyme showed a transient absorption with a maximum at 292 nm which disappeared upon addition of sodium hydroxide. The incubation with UDP-Glc or UDP-GlcUAMe also showed and NAD+-dependent absorption at 292 nm. However, in these cases a strong enhancement of the absorption at alkaline pH and a shift of the absorption maximum to longer wavelength were observed. Upon addition of 0-phenylenediamine to the enzyme incubation with UDP-Glc or UDP-GlcUAe a strong absorption with a maximum at respectively 335 and 315 nm appeared. The results show the transient formation of a 4-keto derivatives in the synthase reaction with the normal substrate UDP-GlcUA. The substrate analogues UDP-Glc and UDP-GlcUAMe can also be oxidized at C-4 by the enzyme in the presence of NAD+ to stable 4-keto derivatives which give rise to a strong absorption at alkaline pH or after reaction with o-phenylenediamine.
D001665:Binding Sites; D001894:Borohydrides; D002262:Carboxy-Lyases / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007659:Ketones; D007700:Kinetics; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D014532:Uridine Diphosphate Glucose / Q000494:pharmacology; D014535:Uridine Diphosphate Glucuronic Acid / Q000494:pharmacology
D001894:Borohydrides; D007659:Ketones; D014535:Uridine Diphosphate Glucuronic Acid; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase; D014532:Uridine Diphosphate Glucose