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) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.