Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

Abstract

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the .beta.-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., and Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 .+-. 0.02 .mu.M at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ > Ca2+ > Co2+ > Mn2+ > Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 .+-. 0.14 .mu.M. 6-Thioorotate has a Ki of 82 .+-. 8 .mu.M which decreases to 3.8 .+-. 1.4 .mu.M in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors. We have synthesized 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate as a ''''transition-state analogue'''' for the reaction catalyzed by dihydroorotase and have found it to be a potent inhibitor with a Ki value of 0.74 .+-. 0.10 .mu.M. By contrast, a cyclic sulfone (5R)-1,1,3-trioxothiomorpholine-5-carboxylate with structural resemblance to the proposed transition state had no inhibitory effect upon dihydroorotase. L-Cysteine and iminodiacetate activate dihydroorotase at low concentrations (0.1 .mu.M) but are effective inhibitors at higher concentrations with apparent Ki values of 550 .mu.M and 200 .mu.M, respectively, for the proposed interaction of a second inhibitor molecule with the enzyme-inhibitor complex. 5-Fluoroorotate is a noncompetitive inhibitor with a dissociation constant for interaction with the free enzyme of 15 .+-. 4 .mu.M, while orotate is a competitive inhibitor with a Ki of 81 .+-. 9 .mu.M.