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., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Amidohydrolases/antagonists & inhibitors , Dicarboxylic Acids/chemical synthesis , Dihydroorotase/antagonists & inhibitors , Pyrimidines/chemical synthesis , Sulfhydryl Compounds/pharmacology , Animals , Binding, Competitive , Cations, Divalent , Cell Line , Cricetinae , Cysteine/analogs & derivatives , Cysteine/pharmacology , Dicarboxylic Acids/pharmacology , Dihydroorotase/isolation & purification , Indicators and Reagents , Kinetics , Magnetic Resonance Spectroscopy , Pyrimidines/pharmacology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The metabolites of the predacious fungus Dactylaria lutea ROUTIEN include the anthraquinone macrosporin (2) and three hydroxylated 1,2,3,4-tetrahydro derivatives of this anthraquinone, altersolanol A (5), altersolanol B (4) and dactylariol (6). The structure and relative configuration of dactylariol are established from spectroscopic studies, and its absolute configuration is proposed as 1R, 2R, 3R by virtue of its co-occurrence with altersolanol B. Dactylarin, suggested by other authors to have the structure (1), is shown to be identical with altersolanol B (4).
