title: Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.
abstract: Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
authors.forename: K
authors.initials: K
authors.lastname: Shostak
authors.forename: M E
authors.initials: ME
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012486:Salmonella typhimurium / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D015032:Zinc / Q000032:analysis
chemical_list: D011742:Pyrimidine Nucleotides; C011569:6-azauridine-5'-monophosphate; D014542:Uridine Monophosphate; D009964:Orotidine-5'-Phosphate Decarboxylase; D015032:Zinc