Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Two constitutive acetyl-CoA acetyltransferases (3-ketothiolases A and B) were purified from Alcaligenes eutrophus. Enzyme A was active with only acetoacetyl-CoA and 3-ketopentanoyl-CoA, whereas enzyme B was active with all the 3-ketoacyl-CoAs (C4−C10) tested. Enzyme A appeared to be a tetramer (Mr 70 000) with identical subunits (Mr 44 000) and enzyme B had a similar Mr of 168 000 (containing Mr 46 000 subunits). Enzymes A and B had isoelectric points of 5.0 and 6.4, respectively. The stoichiometry of the reactions catalysed by each enzyme was confirmed. Km values of 44 µM and 394 µM for acetoacetyl-CoA, and 16 µM and 93 µM for CoA, were determined with enzymes A and B, respectively. Enzymes A and B gave Km values of 1.1 mM and 230 µM, respectively, for acetyl-CoA. The condensation reaction was potently inhibited by CoA in both cases.