Isolation and characterization of the orotidine 5'-monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase.
The multifunctional protein uridine 5'-monophosphate (UMP) synthase catalyzes the final two reactions of the de novo biosynthesis of UMP in mammalian cells by the sequential action of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23). This protein is composed of one or two identical subunits; the monomer weighs of 51,500 daltons. UMP synthase from mouse Ehrlich ascites cells can exist as three distinct species as determined by sucrose density gradient centrifugation: a 3.6 S monomer, a 5.1 S dimer, and a 5.6 S conformationally altered dimer. Limited digestion of each of these three species with trypsin produced a 28,500-dalton peptide that was relatively resistant to further proteolysis. The peptide appears to be one of the two enzyme domains of UMP synthase for it retained only OMP decarboxylase activity. Similar results were obtained when UMP synthase was digested with elastase. OMP decarboxylase activity was less stable for the domain than for UMP synthase; the domain can rapidly lose activity upon storage or upon dilution. The size of the mammalian OMP decarboxylase domain is similar to that of yeast OMP decarboxylase. If the polypeptides which are cleaved from UMP synthase by trypsin are derived exclusively from either the amino or the carboxyl end of UMP synthase, then the size of a fragment possessing the orotate phosphoribosyltransferase domain could be as large as 23,000 daltons which is similar in size to the orotate phosphoribosyltransferase of yeast and of Escherichia coli.
D000818:Animals; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D010430:Pentosyltransferases / Q000302:isolation & purification*; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; C027157:uridine 5'-monophosphate synthase; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D014357:Trypsin; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase