Structural requirements for substrate binding to propionyl-CoA carboxylase

Abstract

Further purification of propionyl-CoA carboxylase (EC 6.4.1.3) from bovine-liver mitochondria is reported. On the basis of the present and earlier investigations it is concluded that binding of acyl-CoA substrates by the carboxylase occurs through the CoA moiety. The relative maximum velocities of propionyl-CoA, propionyl-dephospho-CoA, and propionyl-pantetheine carboxylation were 1.0, 0.29, and 0.014, respectively; the $\text{K}{}_{\mathrm{m}}$ values for the same substrates were 2.6·10⁻⁴, 2.8·10⁻³, and 2.4·10⁻² M, respectively. Enzymatic carboxylation of $\text{propionyl}\text{-N-}\text{acetylcysteamine}$ could not be demonstrated. Coenzyme A and 3′-AMP were found to inhibit the carboxylation reaction competitively with respect to propionyl-CoA, whereas, inhibition by 2′-AMP and 5′-AMP was of a mixed type. The binding of acyl-CoA substrate to the carboxylase appears to involve the 3′-phosphate, adenine, and pantoyl moieties of the subsrate. Propionyl-CoA carboxylase is protected from $\text{p-}\text{chloromercuribenzoate}$ inhibition by propionyl-CoA, whereas, ATP-MgCl₂ facilitates this inhibition.