Identification of lysyl residues located at the substrate-binding site in UDP-glucose pyrophosphorylase from potato tuber: affinity labeling with uridine di- and triphosphopyridoxals.
Uridine di- and triphosphopyridoxals were used to probe the substrate-binding site in potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9). The enzyme was rapidly inactivated in time- and dose-dependent manners when incubated with either reagent followed by reduction with sodium borohydride. The inactivations were almost completely retarded by UDP-Glc and UTP but only slightly by alpha-D-glucose 1-phosphate. The complete inactivation corresponded to the incorporation of about 0.9-1.0 mol of either reagent per mole of enzyme monomer. Both reagents appear to bind specifically to the UDP-Glc-(UTP)-binding site. Structural studies of the labeled enzymes revealed that the two reagents modified the identical set of five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-410), in which Lys-367 was most prominently modified. The ratios of the amounts of labels incorporated into these residues were similar for the two reagents. Furthermore, linear relationships were observed between the residual activities and the amounts of incorporation into each lysyl residue. We conclude that the five lysyl residues are located at or near the UDP-Glc(UTP)-binding site of potato tuber UDP-Glc pyrophosphorylase and that the modification of these residues occurs in a mutually exclusive manner, leading to the inactivation of the enzyme.
Kazuta
Y
Y
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
Omura
Y
Y
Tagaya
M
M
Nakano
K
K
Fukui
T
T
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology
D000345:Affinity Labels; C070826:uridine triphosphopyridoxal; D014530:Uridine Diphosphate; D011732:Pyridoxal Phosphate; C045612:uridine diphosphopyridoxal; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D008239:Lysine; D014544:Uridine Triphosphate