Comparative affinity labeling with reactive UDP-glucose analogues: possible locations of five lysyl residues around the substrate bound to potato tuber UDP-glucose pyrophosphorylase.
By using two reactive analogues of UDP-Glc, uridine di- and triphosphopyridoxals, we have recently probed the substrate-binding site in potato tuber UDP-Glc pyrophosphorylase [EC 2.7.7.9]. In this work, pyridoxal diphospho-alpha-D-glucose was used for the same purpose. This compound is also a reactive UDP-Glc analogue but having its reactive group on the opposite side of the pyrophosphate linkage to those of the above two compounds. The enzyme was rapidly inactivated when incubated with the compound at very low concentrations followed by reduction with sodium borohydride. The inactivation was almost completely prevented by UDP-Glc and UTP. Complete inactivation correspond to the incorporation of 1.0 mol of the reagent per mol of enzyme monomer. The label was found to be distributed in five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-40. All of these results were similar to those obtained previously with the other compounds, suggesting the presence of a cluster of five lysyl residues at or near the substrate-binding site of this enzyme. However, the incorporations of labels into each lysyl residue differed depending on the compounds used. The substrate retarded the incorporations in different manners. Based on the combined results of the present and previous studies, a hypothetical model is presented for the possible locations of the five lysyl residues around the substrate bound to the enzyme. This model is consistent with the kinetic properties of mutant enzymes in which the five lysyl residues were individually replaced by glutamine via site-directed mutagenesis.
Kazuta
Y
Y
Institute of Scientific and Industrial Research, Osaka University.
Tanizawa
K
K
Fukui
T
T
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry*; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives*
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; C032401:pyridoxal(5')diphospho(1)-glucose; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D008239:Lysine; D014532:Uridine Diphosphate Glucose