Molecular cloning, nucleotide sequencing, and affinity labeling of bovine liver UDP-glucose pyrophosphorylase.
A bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2.7.7.9], which catalyzes the reversible uridylyl transfer between glucose 1-phosphate and MgUTP, has been cloned by the use of oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant sequence similarities with the enzymes from potato tuber and a slime mold, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the purified enzyme was incubated with an affinity labeling reagent, uridine triphosphopyridoxal, and then reduced with sodium borohydride. The enzyme was inactivated rapidly and irreversibly by the reagent at low concentrations. The inactivation was almost completely retarded by UDP-glucose and MgUTP. Structural analysis of the labeled enzyme revealed that three lysyl residues, Lys291, Lys357, and Lys396, were modified by the reagent. The three lysyl residues are conserved at the corresponding positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the active-site structure of bovine liver UDP-glucose pyrophosphorylase is very similar to that of the potato tuber enzyme.
Konishi
Y
Y
Institute of Scientific and Industrial Research, Osaka University.
Tanizawa
K
K
Muroya
S
S
Fukui
T
T
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D000345:Affinity Labels; D015345:Oligonucleotide Probes; C070826:uridine triphosphopyridoxal; D011732:Pyridoxal Phosphate; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D008239:Lysine; D014544:Uridine Triphosphate