Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae.

Published

Journal Article

In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.

Full Text

Duke Authors

Cited Authors

  • Langdon, SD; Jones, ME

Published Date

  • September 25, 1987

Published In

Volume / Issue

  • 262 / 27

Start / End Page

  • 13359 - 13365

PubMed ID

  • 3308878

Pubmed Central ID

  • 3308878

International Standard Serial Number (ISSN)

  • 0021-9258

Language

  • eng

Conference Location

  • United States