Purification and properties of 3-hydroxybutyryl-coenzyme A dehydrogenase from Clostridium beijerinckii ('Clostridium butylicum') NRRL B593

Abstract

The enzyme 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii NRRL B593. The identities of 34 of the N-terminal 35 amino acid residues have been determined. The enzyme exhibited a native M(r) of 213,000 and a subunit M(r) of 30,800. It is specific for the (S)- enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 μM, respectively. The maximum velocity of the enzyme was 540 μmol min-1 mg-1 for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as a cosubstrate; however, k(cat)/K(m) for the NADH-linked reaction was much higher than the apparent value for the NADPH-linked reaction. Also, NAD(H)-linked activity was less sensitive to changes in pH than NADP(H)-linked activity was. In the presence of 9.5 μM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 μM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth.