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.
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 transcarbamylase and of N-carbamyl-L-aspartate at the dihydroorotase site must be 2.2-fold and 3.1-fold higher, respectively, than their average concentrations in the bulk solvent. Similar concentrations are predicted by calculation of steady state concentrations from ratios of the rate constants for the three activities. A high local concentration of N-carbamyl-L-aspartate at the third site is also indicated by a 3.6-fold reduction in the transient time for dihydroorotase activity from that predicted. Competition experiments performed with exogenous carbamyl phosphate and N-carbamyl-L-aspartate indicate only partial channeling of these intermediates. The inhibitory effect of N-phosphonacetyl-L-aspartate (PALA), at concentrations up to at least 2.4 microM, upon the aspartate transcarbamylase activity of ME pyr1-3 can be overcome by accumulated carbamyl phosphate. This mechanism for resistance to PALA could be manifest in cells which lack an effective phosphatase activity to hydrolyze carbamyl phosphate. L-Cysteine, a slow acting but potent inhibitor of dihydroorotase in the absence of substrates (Christopherson, R. I., and Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370), also inactivates dihydroorotate when ME pyr1-3 is synthesizing L-5,6-dihydroorotate.
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D002460:Cell Line; D006224:Cricetinae; D004080:Dihydroorotase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008647:Mesocricetus; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D013379:Substrate Specificity
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D001221:Aspartate Carbamoyltransferase; D000581:Amidohydrolases; D004080:Dihydroorotase; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)