Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

• Published in: Biochemistry (Easton) Vol. 28; no. 2; pp. 463 - 470
• Main Authors: Christopherson, Richard I, Schmalzl, Karl J, Szabados, Eve, Goodridge, Richard J, Harsanyi, Michael C, Sant, Melissa E, Algar, Elizabeth M, Anderson, Janet E, Armstrong, Alison
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 1989
• Subjects: Amidohydrolases - antagonists & inhibitors; Animals; Binding, Competitive; Cations, Divalent; Cell Line; Cricetinae; Cysteine - analogs & derivatives; Cysteine - pharmacology; dicarboxylate; Dicarboxylic Acids - chemical synthesis; Dicarboxylic Acids - pharmacology; Dihydroorotase - antagonists & inhibitors; Dihydroorotase - isolation & purification; Indicators and Reagents; Kinetics; Magnetic Resonance Spectroscopy; mercaptan; Pyrimidines - chemical synthesis; Pyrimidines - pharmacology; Structure-Activity Relationship; Substrate Specificity; Sulfhydryl Compounds - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00428a009

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., & 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 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.