PubMed1980
We have studied factors affecting the catalytic activity of dihydroorotase (EC 3.5.2.3), purified as part of a multienzymatic protein which contains carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase (ME pyr1-3) and which initiates de novo pyrimidine biosynthesis in mouse Ehrlich ascites carcinoma. The apparent Km value for N-carbamyl-L-aspartate increases by 2 orders of magnitude as the pH increases from 7.0 to 8.3, consistent with equilibration of dihydroorotase (E) between four states of protonation (E in equilibrium EH in equilibrium EH2 equilibrium EH3), where EH3 is the only catalytically active form of dihydroorotase for the biosynthetic reaction, having a Km for N-carbamyl-L-aspartate of 30 micro M. The apparent Km for L-dihydroorotate shows a converse dependence upon pH, remaining relatively constant at alkaline pH and increasing progressively as the pH is decreased below 7.0. These data are consistent with the above model if E and EH are catalytically active for the degradative reaction, both having Km values of 4.4 micro M for L-5,6-dihydroorotate. The D isomers of carbamylaspartate and dihydroorotate are also substrates for dihydroorotase. At pH 7.33, the apparent Km values for N-carbamyl-L-aspartate and N-carbamyl-D-aspartate are 247 and 204 micro M, respectively, but the Vmax for N-carbamyl-D-aspartate is only 1.7% of that obtained with N-carbamyl-L-aspartate. Orotate and a series of 5-substituted derivatives are competitive inhibitors of dihydroorotase. At pH 7.27, the apparent Ki for orotate using N-carbamyl-L-aspartate as substrate is 170 micro M and with L-5,6-dihydroorotate as substrate, the apparent Ki value is 9.6 micro M, suggesting that the enzyme exists in different forms in the presence of each substrate. Dihydroorotase is inhibited in a time-dependent manner by 50 mM L-cysteine and the presence of N-carbamyl-L-aspartate or L-5,6-dihydroorotate protects against this ultimately complete inactivation. 2-Mercaptoacetate, 2-mercaptoethylamine, 3-mercaptopropionate, and L-2,3-diaminopropionate have a similar although less potent inhibitory effect. To account for the data obtained, we propose a model for the equilibria existing between various protonated forms of dihydroorotase which is consistent with the pH dependencies of the apparent Km values observed and the Vmax values observed previously (Christopherson, R.I., and Jones, M.E. (1979) J. Biol. Chem. 254, 12506-12512). In addition, a catalytic mechanism is presented for the interconversion of N-carbamyl-L-aspartate and L-5,6-dihydroorotate.