Flavoenzymes Involved in Pyrimidine Oxidation and Reduction.

Abstract

Pyrimidines are chemical compounds found throughout nature. They occur in DNA, RNA, antibiotics, and a myriad of other natural products. One of the many chemical alterations found to occur during the biosynthesis, degradation, and modification of pyrimidines is that of carbon-carbon bond reduction or oxidation. The majority of the enzymes known to perform this reaction are flavo-enzymes, which catalyze either the reduction or oxidation of the carbon-carbon bond at the C5 and C6 positions of the pyrimidine ring. Enzymes from two of these classes are studied here, the dihydroorotate dehydrogenases and the dihydrouridine synthases. Dihydroorotate dehydrogenases catalyze the formation of a carbon double-bond in the biosynthesis of uracil, by oxidizing dihydroorotate to orotate. The kinetic mechanism for the dihydroorotate dehydrogenase from Enterococcus faecalis was investigated. The kinetic rate constants for the oxidation of dihydroorotate, the off rate for the product orotate, and the oxidative rate constant for the oxidation by fumarate were determined at pH 8.5, 4 oC. The steady state kinetics of the reaction were also determined. These data indicate that the oxidation of the enzyme by fumarate is the rate limiting step for the enzyme. The binding constants for the ligands fumarate, succinate, orotate, and dihydroorotate were determined as well as the binding constants for the ligands 3,4-dihydroxybenzoate and 3,5-dihydroxybenzoate. The effect of pH upon the reaction was investigated. The results of these experiments indicate a variable pKa affecting the oxidative and reductive half-reactions. This pK¬a seems to be affected by the presence of different substrates in the active site during each half-reaction. The pKa ¬changes from 8.3 for the reductive half-reaction to 7.5 for the oxidative half-reaction. The origin of the pKa is most likely an active site cysteine. A second pKa above 12 was also observed, affecting the binding of the ligands fumarate, orotate, and dihydroorotate to the enzyme. This pKa is attributed to the active site lysine, lysine 55. Crystal structures of this enzyme with dihydroorotate, L-malate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, and orotate were obtained.