The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols

Abstract

1 Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans was inactivated by incubation with n‐alkanols at 37°C. The concentration of the alcohol required for complete inactivation decreased with increasing chain length; e.g. 2 M ethanol was as potent as 2 mM hexanol or 0.5 mM decanol. The data indicate a binding of the alcohol to the enzyme with an energy of about 4 kJ/methylene group. 2 Sodium ions prevented the inactivation (50% at 30 mM NaCl). K⁺, NH₄⁺, Cs⁺ and Mg²⁺ had no influence, whereas Li⁺ was ten times less effective than Na⁺. 3 The enzyme was cleaved during the inactivation into a soluble part, consisting of the α (M_r 1 20 000) and β polypeptide chains (60 000), whereas the hydrophobic γ chain (30 000) precipitated. 4 The soluble part catalysed the sodium‐ion‐independent but avidin‐sensitive glutaconyl‐CoA/crotonyl‐CoA exchange as measured with the substrates [3‐³H]crotonyl‐CoA and unlabelled glutaconate and with glutaconate CoA‐transferase as auxiliary enzyme. 5 In the presence of free biotin or its methyl ester the soluble part catalysed the formation of crotonyl‐CoA from glutaconyl‐CoA (apparent K_m for biotin 40 mM, V_max 1% of the native decarboxylation reaction). This apparent reactivation was most likely caused by the carboxylation of free biotin. 6 Based on these and other observations the following functions may be assigned to the different polypeptide chains of glutaconyl‐CoA decarboxylase: biotin carrier (α), carboxytransferase (β) and carboxylase, the actual sodium pump (γ).