Orotate phosphoribosyltransferase and orotidylate decarboxylase from Crithidia luciliae: subcellular location of the enzymes and a study of substrate channeling.
Orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) have been found to be particulate in the kinetoplastid protozoan, Crithidia luciliae. Sucrose density centrifugation indicated that these two enzymes are associated with the glycosome, a microbody which appears to be unique to the Kinetoplastida and which contains many of the glycolytic enzymes. The particulate location of OPRTase and ODCase was considered to be favorable for channeling of orotidine-5'-monophosphate (OMP), the product of the first enzyme and substrate for the second. The degree of channeling was determined by double radioactively labeled experiments designed to determine the relative efficiency of endogenous and exogenous OMP as substrates of ODCase. The efficiency of channeling was high, with an approximate 50-fold preference for endogenous OMP. By comparison, the degree of channeling for the yeast enzymes, which are soluble and unassociated, was less than 2-fold. The OPRTase-ODCase enzyme complex was solubilized using Triton X-100 in the presence of dimethyl sulfoxide, glycerol, and phosphoribosyldiphosphate. The percentage recovery of the overall enzyme activity was approximately 20%. The degree of channeling was reduced by approximately 10-fold for the solubilized complex. The Km for OMP changed from 7.5 (+/- 1.8) to 1.6 (+/- 0.3) microM in the ODCase reaction. There was no alteration in the Km for orotate in the OPRTase reaction.
D000818:Animals; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D003421:Crithidia / Q000201:enzymology*; D009962:Orotate Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D010430:Pentosyltransferases / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000201:enzymology
D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase