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Acyl Carrier Protein

XX. CHEMICAL SYNTHESIS AND CHARACTERIZATION OF ANALOGUES OF ACYL CARRIER PROTEIN

From the ¹ From the Washington University School of Medicine, Departments of Biological Chemistry and of Physiology and Biophysics, St. Louis, Missouri 63110

A number of analogues of Escherichia coli apo-acyl carrier protein (apo-ACP) have been synthesized by the solid phase method. A standard isolation procedure was used for all analogues. This involved cleavage from the solid support and deprotection by treatment of the peptide-resin with hydrogen bromide and trifluoroacetic acid. The product was then purified by gel filtration. When necessary the deprotection of N-nitroarginine was achieved by hydrogenation. The prosthetic group, 4'-phosphopantetheine, was introduced enzymatically with holo-ACP synthetase, and the holoprotein was purified by ion exchange chromatography. The product was then assayed for biological activity with the enzymes malonyl coenzyme A-ACP transacylase and -ketoacyl-ACP synthetase and with antiserum prepared against native ACP.

The following analogues were synthesized: [Arg(NO₂)⁶]-apo-ACP-(1-74); apo-ACP-(2-74); apo-ACP-(3-74); [Arg(NO₂)⁶,Nle^{8, 9, 18}]-apo-ACP-(1-74); [Arg(NO₂)⁶,Nle⁴⁴]-apo-ACP-(1-74); [Nle^{6, 44}]-apo-ACP-(1-74); [Arg(NO₂)⁶, Nle^{8, 9, 18, 44}]-apo-ACP-(1-74); [Arg(NO₂)⁶,Nle^{8, 9, 18, 44}]-apo-ACP-(2-74); [Arg(NO₂)⁶,Nle^{8, 9, 18, 44}]-apo-ACP-(3-74); [Arg(NO₂)⁶,Nle^{8, 9, 18, 44}]-apo-ACP-(4-74); [Arg(NO₂)⁶, Nle^{8, 9, 18, 44}]-apo-ACP-(5-74); [Arg(NO₂)⁶,Nle^{8, 9, 18, 44}]-apo-ACP-(6-74); [Nle^{8, 9, 18, 44}]-apo-ACP-(7-74); and [Arg(NO₂)⁶, Nle^{8, 9, 18, 44}]-apo-ACP-(1-61). A study of the activity of these derivatives indicated that the single methionine at position 44 and the three lysines at positions 8, 9, and 18 play a nonessential role in the biological function of ACP. The single arginine at position 6 is more critical, for although the N-nitroarginine derivative was active, substitution of norleucine for arginine gave an analogue which was inactive with malonyl-CoA-ACP transacylase. While a large part of the COOH terminus was not essential, since the fragment 1 through 61 retained significant activity, the NH₂ terminus was much more critical and the omission of more than 3 residues caused a significant reduction in the biological activity of the protein.

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