Acyl Carrier Protein: STRUCTURAL REQUIREMENTS FOR FUNCTION IN FATTY ACID BIOSYNTHESIS

Abstract

Abstract The mechanism of the reactivity of the enzymes of fatty acid synthesis from Escherichia coli with thioester substrates of acyl carrier protein has been investigated by structural modification of this protein. Carboxypeptidase A treatment of acyl carrier protein releases the carboxyl-terminal residue, alanine, and 0.3 residue each of histidine and glutamine. Carboxypeptidase A-treated acyl carrier protein remains fully active as assayed in the malonyl-coenzyme A CO2-exchange reaction of fatty acid synthesis. Limited acid hydrolysis of acyl carrier protein at pH 2.0, 100°, results in the preferential cleavage and release of aspartic acid residues and rapidly inactivates the protein. After a single aspartic acid residue has been released, 80% of acyl carrier protein activity has been lost. Treatment of acyl carrier protein for 80 min at pH 2.0, 100°, results in the release of 0.39 mole of aspartic acid per mole of acyl carrier protein. Following this treatment a single peptide was isolated in large yield. This peptide comprised three-fourths of the molecule and contained the intact carboxyl-terminal end of acyl carrier protein. The amino terminus of this peptide was serine, and the peptide was inactive in the malonyl-CoA CO2-exchange reaction. After tryptic hydrolysis of acyl carrier protein a peptide containing 42 amino acid residues was isolated. This peptide contained the intact prosthetic group. It had aminoterminal glutamic acid and the carboxyl-terminal sequence (Leu,Val)-Met-Ala-Lys, and was unable to replace acyl carrier protein, either in the malonyl-CoA CO2-exchange reaction or in long chain fatty acid synthesis. Malonyl tryptic peptide was unable to replace malonyl acyl carrier protein in the β-ketoacyl acyl carrier protein synthetase reaction, thus explaining the inability of this peptide to function in long chain fatty acid synthesis. Acetoacetyl tryptic peptide was reduced slowly in the presence of β-ketoacyl acyl carrier protein reductase with a Michaelis constant of 1.7 x 10-3 m and a maximum velocity of 0.21 µmole of reduced triphosphopyridine nucleotide oxidized per min per mg, compared to values of 6.6 x 10-5 m and 1.36 µmoles of TPNH oxidized per min per mg for acetoacetyl acyl carrier protein. This difference suggests that the site which confers the high reactivity of the acyl carrier protein substrate is missing in the tryptic peptide. The existence of such a site is indicated by the fact that free acyl carrier protein competitively inhibited (Ki = 2.2 x 10-4 m) the reduction of acetoacetyl acyl carrier protein catalyzed by β-ketoacyl acyl carrier protein reductase. The tryptic peptide at concentrations up to 5 x 10-4 m did not inhibit the reduction of acetoacetyl acyl carrier protein. These experiments suggest that acyl carrier protein interacts with β-ketoacyl acyl carrier protein reductase at a site different from the prosthetic group-active site interaction and that this site has been altered or lost upon tryptic digestion of acyl carrier protein. Further primary structure of acyl carrier protein is presented.