Purifications and properties of orotidine-phosphorolyzing enzyme and purine nucleoside phosphorylase from Erwinia carotovora AJ 2992.
An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1,2,4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68,000 +/- 2,000, and seemed to be a dimer. The optimal temperatures and pH values were 60 degrees C and 6.0 for orotidine phosphorolysis, and 70 degrees C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40 degrees C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58,000 +/- 2,000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40 degrees C. The PNPase was completely inactivated by p-chloromercuribenzoate.
Shirae
H
H
Central Research Laboratories of Ajinomoto Co., Ltd., Kawasaki, Japan.
Yokozeki
K
K
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D016973:Pectobacterium carotovorum / Q000201:enzymology*; D010710:Phosphates / Q000494:pharmacology; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012254:Ribavirin / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014543:Uridine Phosphorylase / Q000302:isolation & purification* / Q000378:metabolism
D008670:Metals; D010710:Phosphates; D012254:Ribavirin; D011683:Purine-Nucleoside Phosphorylase; D014543:Uridine Phosphorylase