Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.
Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.
Kazuta
Y
Y
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
Tagaya
M
M
Tanizawa
K
K
Fukui
T
T
D000345:Affinity Labels; D001665:Binding Sites; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D011198:Solanum tuberosum / Q000201:enzymology; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; C020797:pyridoxal diphosphate; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase