A reexamination of the substrate utilization of 2-thioorotidine-5'-monophosphate by yeast orotidine-5'-monophosphate decarboxylase.
A potential alternate substrate for orotidine-5'-monophosphate decarboxylase, 2- thio-orotidine-5'-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155-12161). Characterization of the product was confirmed by mass spectrometry, (31)P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10(-7) from the k(cat) for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 microM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase.
Smiley
J A
JA
Department of Chemistry, Youngstown State University, OH 44555, USA. jasmiley@cc.ysu.edu
Hay
K M
KM
Levison
B S
BS
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003653:Decarboxylation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism*
C445705:2-thioorotidine-5'-monophosphate; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase