In acetone/butanol-producing clostridia, the metabolic intermediate acetoacetyl-CoA can be directed toward butyrate or butanol formation by the reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase, or toward acetone formation by the reaction catalyzed by acetoacetate:acetate/butyrate CoA-transferase. 3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35 or 1.1.1.157) has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii strain NRRL B593. The identities of 34 of the 35 N-terminal 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 mg) for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as cosubstrate; however, NAD(H) appeared to be the physiological substrate. 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.

Acetoacetate:acetate/butyrate CoA-transferase (EC 2.8.3.9) has been purified 308-fold to apparent homogeneity from the same organism. The enzyme exhibited a native M_r of 89,100. The subunits of the enzyme were separated by preparative SDS-PAGE, and exhibited M, values of 28,400 and 25,200. The identities of the 34 N-terminal amino acids of the large subunit and 38 of the 39 N-terminal amino acids of the small subunit were determined. The N-terminal region of the two subunits showed significant similarity with several other CoA transferase enzymes. Michaelis constants for butyrate and acetoacetyl-CoA were 11.7 mM and 107 µM, respectively, while those for acetate and acetoacetyl-CoA were 424 mM and 118 µM, respectively. The value of k_cat/K_m was approximately 100 times higher with butyrate than with acetate.

Implications of the properties of these two enzymes for the acetone-butanol fermentation are discussed, and a model for the induction of the enzymes responsible for solvent production is suggested.