[27] Preparative enzymatic synthesis of acyl-acyl carrier protein

This chapter discusses the preparative enzymatic synthesis of acyl-acyl carrier protein. Fatty acids are synthesized de novo as thioesters of acyl carrier protein (ACP), and long-chain acyl-ACPs serve as substrates for the snglycerol-3-phosphate acyltransferase in Escherichia coli. The most widely used chemical procedure results in preparations of acyl-ACP possessing highly variable biological activity, due to acetylation of the ɛ-amino groups of the four lysine residues and the NH2-terminal serine. Acetylation has been shown to result in a dramatic loss of secondary structure and alteration of the biological activity of ACP-SH and of acyl-ACP. It is found that when large amounts of acyl-ACP are being prepared it is useful to recover acyl-ACP synthetase from the incubation mixtures for use in further syntheses. If acyl-ACP synthetase is not going to be recovered, this step can be omitted and the incubation mixture applied directly to DEAE-cellulose. Acyl-ACP is much more stable to base-catalyzed hydrolysis than the analogous acyl-CoA 7. This property allows experiments with acyl-ACP at pH values up to 10.0 to be performed without significant hydrolysis occurring. The presence of the acyl moiety on ACP also stabilizes the protein to pH-induced hydrodynamic expansion, permitting the separation of ACP from acyl-ACP on gel filtration columns.