Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei.
Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.
Avraham
Y
Y
Department of Bacteriology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Grossowicz
N
N
Yashphe
J
J
D019325:3-O-Methylglucose; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008757:Methylglucosides / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000302:isolation & purification*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013939:Thymidine Phosphorylase / Q000302:isolation & purification* / Q000378:metabolism; D014543:Uridine Phosphorylase / Q000302:isolation & purification* / Q000378:metabolism
D008757:Methylglucosides; D019325:3-O-Methylglucose; D010430:Pentosyltransferases; D014543:Uridine Phosphorylase; D013939:Thymidine Phosphorylase