Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase.
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)
Christopherson
R I
RI
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Schmalzl
K J
KJ
Szabados
E
E
Goodridge
R J
RJ
Harsanyi
M C
MC
Sant
M E
ME
Algar
E M
EM
Anderson
J E
JE
Armstrong
A
A
Sharma
S C
SC
D000581:Amidohydrolases / Q000037:antagonists & inhibitors*; D000818:Animals; D001667:Binding, Competitive; D002413:Cations, Divalent; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D003998:Dicarboxylic Acids / Q000138:chemical synthesis* / Q000494:pharmacology; D004080:Dihydroorotase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011743:Pyrimidines / Q000138:chemical synthesis* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D002413:Cations, Divalent; D003998:Dicarboxylic Acids; D007202:Indicators and Reagents; D011743:Pyrimidines; D013438:Sulfhydryl Compounds; D000581:Amidohydrolases; D004080:Dihydroorotase; D003545:Cysteine