PMID: 6108323Dec 10, 1980Paper

The overall synthesis of L-5,6-dihydroorotate by multienzymatic protein pyr1-3 from hamster cells. Kinetic studies, substrate channeling, and the effects of inhibitors.

The Journal of Biological Chemistry
R I Christopherson, M E Jones

Abstract

In mammals, a trifunctional protein (ME pyr1-32) synthesizes L-5,6-dihydroorotate in three sequential reactions catalyzed by carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). 14C-labeled HCO3- has been used as a precursor for the synthesis of L-5,6-dihydroorotate by purified ME pyr1-3, and when this product is converted enzymatically to orotidine 5'-monophosphate, the concentrations of the two intermediates of ME pyr1-3, carbamyl phosphate, and N-carbamyl-L-aspartate, reach steady state concentrations of approximately 0.20 microM and 7.1 microM, respectively. At pH 7.4 in the presence of 0.1 mM 5-phosphoribosyl 1-pyrophosphate and 10% (v/v) glycerol, pure ME pyr1-3 has a Michaelis constant for HCO3- of 0.61 mM and a maximal specific activity of 329 pmol of L-5,6-dihydroorotate synthesized/min/microgram, equivalent to a turnover number of 65.8 mol min-1 (mol of subunit)-1. Consideration of the Km and Vmax values of aspartate transcarbamylase and dihydroorotase determined under the same conditions as the overall rate of synthesis of L-5,6-dihydroorotate by ME pyr1-3, indicates that the local concentrations of carbamyl phosphate at the active site of aspartate tra...Continue Reading

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.