Direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate decarboxylase.
New sensitive and direct spectrophotometric assays for orotate phosphoribosyltransferase and orotidylate-5'-monophosphate (OMP) decarboxylase are described. The assays utilize a thioketone derivative of orotate (4-thio-6-carboxyuracil) which is converted into 4-thio-OMP by the transferase in the presence of phosphoribosyl pyrophosphate. 4-Thio-OMP is subsequently decarboxylated to 4-thio-UMP by OMP decarboxylase. A novel, efficient synthesis of thioorotate is described. Unlike the natural substrates, the interconversion of the thioketone derivatives yields large spectral changes in the near-visible absorption region. Orotate phosphoribosyltransferase is assayed at 333 nm with a molar extinction coefficient of 10,300 M-1 cm-1 for the conversion of thioorotate to either 4-thio-OMP or 4-thio-UMP. Orotidylate decarboxylase is assayed at 365 nm with a molar extinction coefficient of 3350 M-1 cm-1 for the conversion of 4-thio-OMP to 4-thio-UMP. Another advantage of these substrates is that they bind less tightly to orotate phosphoribosyltransferase and OMP decarboxylase than orotate or OMP, respectively. Thus, the initial rates of substrate conversion to product are readily measurable near the Km values for the thioketone substrates. The ability to follow the reactions directly permits the rapid determination of Km values for the thioketone substrates and Ki values for inhibitors of the enzymes.
Shostak
K
K
Department of Biochemistry, University of North Carolina, Chapel Hill 27599.
Christopherson
R I
RI
Jones
M E
ME
D005658:Fungi / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009962:Orotate Phosphoribosyltransferase / Q000378:metabolism*; D009963:Orotic Acid / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods*
D009963:Orotic Acid; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase