Portland State University. Department of Biology
Date of Award
Master of Science (M.S.) in Biology
1 online resource (viii, 72 leaves)
Dihydroorotate dehydrogenase, Dehydrogenase
Dihydroorotate (DRO) dehydrogenase catalyzes the oxidation of DHO to orotate in the pyrimidine biosynthetic pathway. This enzyme was originally isolated from a bacterium, Zymobacterium oroticum, which would ferment orotate as a sole source of energy. This adaptive catabolic enzyme, which catalyzes the reduction of orotate to DRO in an efficient pyridine nucleotide-linked reaction, has been extensively studied by several workers. Until recently, no study has been carried out on the enzyme which catalyzes the reaction in the biosynthetic direction. Preliminary studies have shown that the biosynthetic enzyme in Esherichia coli and a pseudomonad is not capable of reducing orotate to DRO by a pyridine nucleotide-linked reaction. These results suggested that there may be significant differences between the catabolic and biosynthetic enzymes. In the present study biosynthetic DHO dehydrogenase from Lacto-bacillus bulgaricus was investigated on the basis of physical and kinetic properties in order to compare the enzyme with the extensively studied catabolic enzyme. The stoichiometry exhibited by the DHO oxidase activity of the biosynthetic enzyme and the absorption spectrum suggest that biosynthetic DHO dehydrogenase is a flavoprotein. Thin layer chromatography of the flavins extracted from the enzyme and reactivation of apoenzymes specific for flavin mononucleotide or flavin adenine dinucleotide have shown that the enzyme contains flavin mononucleotide. The demonstration of enzyme-catalyzed sulfite autoxidation suggested that iron is present and is involved in electron transport. Inhibitor studies have shown that the enzyme contains sulfhydryl groups and the inactivation of such groups halts internal electron transport early in the sequence. Kinetic studies were carried out including the determination of the Km for dihydroorotate, Ki for orotate, and the pH optimum. The kinetic behavior of the enzyme in the presence of various inhibitors suggest that the essential sulfhydryl groups reside at or near the active site. Ammonium sulfate was found to enhance the activity of the enzyme. Evidence presented suggested that this phenomenon is probably an unspecific anion effect in which the rate constant for the breakdown of the enzyme substrate complex is directly affected. A possible scheme of the internal electron transport of biosynthetic DHO dehydrogenase was presented, using the data from this thesis and additional evidence from studies carried out by other workers on similar enzymes. A summary of the physical and kinetic properties of biosynthetic and catabolic DHO dehydrogenase was presented and a detailed comparison between the two enzymes made.
Taylor, Craig David, "Physical and kinetic properties of dihydroorotate dehydrogenase from Lactobacillus bulgaricus" (1969). Dissertations and Theses. Paper 62.