UDP-apiose/UDP-xylose synthase. Subunit composition and binding studies.
The UDP-apiose/UDP-xylose synthase from cell suspension cultures of parsley has been purified 1400-fold by an improved method. The ratio of apiose to xylose formed from UDP-D-glucuronic acid (UDP-GlcUA) remained constant throughout the purification procedure. Dodecylsulfate-gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is  composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE-cellulose at different pH-values, and on omega-aminoalkyl-Sepharose, and by gel filtration on Acrylex P-100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N-terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids. Separation of the two proteins was achieved on DEAE-cellulose in the presence of urea. After dialysis only the 86000-Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000-Mr protein enzyme stability was decreased drastically. By equilibrium dialysis it was found that 0.5 mol UDP-GlcUA are bound per mole of 86000-Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4-keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C-3 of the 4-ulose intermediate prior to ring contraction to apiose was found.
D002262:Carboxy-Lyases / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D014508:Urea; D014535:Uridine Diphosphate Glucuronic Acid
D046911:Macromolecular Substances; D009243:NAD; D014535:Uridine Diphosphate Glucuronic Acid; D014508:Urea; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase