Propionyl carboxylase purified from bovine liver mitochondria catalyzes the carboxylation of 992 micromoles of propionyl-CoA per hour per milligram of protein. Relative carboxylation rates for acetyl-, propionyl-, butyryl-, and valeryl-CoA remain constant during purification. The carboxylase is inhibited by PCMB, N-ethylmaleimide, and iodoacetamide; and the inhibition by PCMB can be almost completely reversed by GSH. The K_m values for acetyl-CoA, propionyl-CoA, butyryl-CoA, valeryh-CoA, propionyl pantetheine, ATP, and HCOj were determined. The K_m values for the aeyl-CoA derivatives are approximately the same while there is a 200-fold difference between the V_m values for propionyl-CoA and valeryl-CoA. Coenzyme A and valeryl-CoA, but not propionyl pantetheine were found to be competitive inhibitors of propionyl carboxylase. The apparent equilibrium constant for the enzymatic propionyl-CoA carboxylation reaction at pH 8.15 and 37°c is 8.1 x 10^-3 and the Δ F°₃₁₀ calculated from this constant is 2970 calories per mole. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / Oxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC 1.2.3.4 OxOx). In this work, wild-type (WT) Bacillus subtilis OxDC and a series of variants with mutations on conserved residues were characterized to investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided information about the mechanism. The Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen and oxalate. Mutations on the second shell residues in the N-terminal metal binding site affected the enzyme activity properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the previously proposed mechanism in which OxDC and the OxDC/OxOx chimeric variant share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing the interaction between the N-terminal and C-terminal domains.