Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies

Biochemistry. 1992 Dec 8;31(48):12155-61. doi: 10.1021/bi00163a026.


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)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Catalysis
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Orotidine-5'-Phosphate Decarboxylase / antagonists & inhibitors
  • Orotidine-5'-Phosphate Decarboxylase / chemistry
  • Orotidine-5'-Phosphate Decarboxylase / metabolism*
  • Pyrimidine Nucleotides / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Salmonella typhimurium / enzymology
  • Sequence Homology, Amino Acid
  • Spectrum Analysis
  • Substrate Specificity
  • Uridine Monophosphate / analogs & derivatives
  • Uridine Monophosphate / metabolism
  • Zinc / analysis


  • Pyrimidine Nucleotides
  • 6-azauridine-5'-monophosphate
  • Uridine Monophosphate
  • Orotidine-5'-Phosphate Decarboxylase
  • Zinc