The thiolase involved in biosynthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera generates an acetyl-enzyme species during catalysis. Up to 0.86 [14C] acetyl eq/subunit of this homotetrameric enzyme is accumulated by acid precipitation in the presence of [14C]acetyl-CoA. Gel filtration of the same solutions produced only 7% acetyl-enzyme suggesting hydrolytic lability of the acetyl-enzyme during the 10-min isolation at 4 degrees C. In an effort to identify active site residues which may function as basic groups to deprotonate at C-2 of acetyl-CoA to generate the required nucleophilic equivalent in carbon-carbon bond formation, we have prepared and tested haloacetyl-thioesters, oxoesters, and amides in the panthetheine pivalate series (Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., and Walsh, C. T. (1987) J. Biol. Chem. 262, 82-89). The [14C]bromoacetyl-oxoester alkylatively inactivates thiolase irreversibly with stoichiometric incorporation of four labels/tetramer. Determination of amino acid composition of the radiolabeled tryptic peptide indicated trapping of Cys-89 (Peoples, O. P., Masamune, S., Walsh, C. T., and Sinskey, A. J. (1987) J. Biol. Chem. 262, 97-102), the same residue modified by iodoacetamide. When the bromoacetyl-thioester was used, inactivation was pH-dependent. The data are consistent with the competition of two processes, acylation, and alkylation. Direct (rather than secondary) alkylation of thiolase by the inactivator accounts for the significant 14C incorporation into thiolase with the thioester labeled with [14C] in the pantetheine pivalate moiety. It appears likely that the haloacetyl analogs described herein should be generally useful for affinity labeling other enzymes using acetyl-CoA as a substrate.
