Radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes.
A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. Neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated 14CO2 is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement of Km values for both substrates. IDCase activity is found to be significantly higher in N. crassa strains lacking uc-1, a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimated Ki value that is too low for accurate determination.
Smiley
J A
JA
Department of Chemistry, Youngstown State University, Youngstown, Ohio, 44555, USA.jasmiley@cc.ysu.edu
Angelot
J M
JM
Cannon
R C
RC
Marshall
E M
EM
Asch
D K
DK
D000645:Ammonium Sulfate / Q000737:chemistry; D002245:Carbon Dioxide / Q000737:chemistry; D002250:Carbon Radioisotopes / Q000032:analysis; D002262:Carboxy-Lyases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D013936:Thymidine / Q000378:metabolism*; D014498:Uracil / Q000031:analogs & derivatives / Q000494:pharmacology
C117583:2-thioisoorotic acid; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D002245:Carbon Dioxide; D014498:Uracil; C057449:5-nitrouracil; D009963:Orotic Acid; D002262:Carboxy-Lyases; C117582:isoorotate decarboxylase; D000645:Ammonium Sulfate; D013936:Thymidine