Investigation into two mechanisms of unusual decarboxylases : [alpha]-amino-[beta]-carboxymuconate [epsilon]-semialdehyde decarboxylase and orotidine 5'-monophosphate decarboxylase

Abstract

ACMSD and ODCase are two different decarboxylase enzymes with mechanisms that deviate from the typical mechanisms found for these types of enzymes. Studying these enzymes may help elucidate these extraordinary mechanisms in which the substrate is converted to a product with carbon dioxide. Our group intends on producing recombinant mouse ACMSD for characterization and insight into ACMSD's mechanism. Because ACMS is unstable, the enzyme preceding ACMSD, 3-hydroxyanthranilate oxygenase (3-HAO) was used to produce the substrate. Alternate substrates fro 3-HAO were tested in order to find a more stable substrate or inhibitor for ACMSD. ACMSD's mechanism is proposed to involve a Michael addition where a nucleophilic attack on the substrate is used to stabilize the carbanion formed. The mechanism proposed for ODCase involves a zwitterionic intermediate using the active site lysine (Lys73 in E. coli) to protonate O2 on the pyrimidine ring of orotidine 5'-monophosphate (OMP) an then subsequent decarboxylation at C6. Using a semi-enzymatic synthesis of [18O2, 13C-carboxyl] OMP, analysis of ODCase can be performed by measuring carbon isotope effects for the decarboxylation reaction of natural abundance and specifically labeled substrate. Measurement of isotope effects involves collecting carbon dioxide produced by the reaction of OMP and ODCase and analysis of the CO2 using an isotope ratio mass spectrometer. If the isotope effects produce significantly different values with the natural abundance and specifically labeled substrates, this would help support the mechanism proposed.