OROTIDYLATE DECARBOXYLASE - INSIGHTS INTO THE CATALYTIC MECHANISM FROM SUBSTRATE-SPECIFICITY STUDIES

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. The decarboxylation of orotidylate by orotidylate decarboxylase is unusual because, unlike other decarboxylases, the reaction requires no known cofactors. In addition to the studies above that do not support the formation of an enzyme-substrate addition product, we carried out atomic absorption and chemical tests to see if the enzyme contained tightly bound zinc; none was found. However, spectral studies to explore the interaction between the enzyme and 6-aza-UMP, a potent inhibitor, again rule out a mechanism involving loss of the double bond between positions 5 and 6 but are consistent with a tautomeric shift from the 2-keto, 4-keto form of the free inhibitor to a 2-enol, 4-keto form once the inhibitor is bound to the enzyme.