Charakterisierung der Acetyltransferase in der Fettsaäresynthetase aus Hefe
Authors: Ziegenhorn Joachim , Niedermeier Rosa , Nüssler Christa , Lynen Feodor 
Published: March 2005

The influence of iodoacetamide and N‐ethylmaleinimide on the activity of the acetyl transferase component of fatty acid synthetase from baker's yeast was studied using [14C]acetyl‐CoA and pantetheine as substrates. No inhibition by either of the blocking agents was observed. This, together with previous information, suggests, that a non‐sulfhydryl acceptor group is involved in the catalytic process of acetyl transfer from acetyl‐CoA to the acyl carrier protein component of the multienzyme complex.

To identify this carrier group in the active site of its acetyl transferase, fatty acid synthetase was treated with [14C]acetyl‐CoA after first blocking the free SH‐groups of the complex with Ellman's reagent. In this way a radioactive acetyl‐enzyme was formed, which contained approximately 3 acetyl residues per molecule of protein (molecular weight 2.3 × 106) in covalent non‐thioester linkage.

Peptic digestion of the [14C]acetyl‐enzyme yielded a complex mixture of peptides which could be separated into 16 different radioactive fractions after extended chromatographic and electrophoretic procedures. Amino acid analysis established the main constituents of this mixture to be a nonapeptide and an undecapeptide of the composition [14C]Ac(Lys,His,Thr,Ser,Glx,Gly3,Ala) and [14C]Ac(Lys,His,Thr,Ser,Glx,Gly4,Ala,Leu). Alanine occupied the N‐terminal position in the nonapeptide.

Tryptic cleavage of the [14C]acetyl‐enzyme liberated a decreased number of peptides, which made their separation much easier. The digestion experiments carried out at pH 6.9–7.0 in order to retain intact alkali‐labile acetyl acceptor bonds gave rise in high yields to a [14C]acetyl‐heptapeptide and a [14C]acetyl‐octapeptide, which could be isolated in pure form. Using stepwise degradation by carboxypeptidase A the structures of these peptides were shown to be [14C]Ac‐Ser‐Gln‐Gly‐Leu‐Val‐Thr‐Ala and[14C]Ac‐Ser‐Gln‐Gly‐Leu‐Val‐Thr‐Ala‐Val.

The rates of base‐catalyzed hydrolysis of [14C]acetyl‐enzyme, of the peptic [14C]acetyl‐peptides and of the model compound Glp‐Ser([14C]Ac)‐Gly in 8 M urea were compared. All the compounds reacted at the same rate. This provided evidence that the acetyl group is bound in an ester linkage to a hydroxyl group of a serine residue in the denatured [14C]acetyl‐enzyme and in the peptic [14C]acetyl‐peptides.

The N‐terminal position of the [14C]acetyl group in the tryptic peptides proved to be the result of an O→N‐acetyl shift at the serine residue, which spontaneously followed the liberation of the peptides by trypsin.

After pretreatment of the [14C]acetyl‐enzyme with carboxypeptidase A in 6 M urea followed by digestion with trypsin no decrease in the amount of the N‐[14C]acetyl‐heptapeptide and the N‐[14C]acetyl‐octapeptide formed could be detected. From this observation it was concluded, that the peptides, although free of basic amino acids, are not derived from the C‐terminal ends of polypeptide chains of the enzyme. Their formation was due to the action of trypsin and possibly to the presence of proteolytic contaminations in the commercial diphenylcarbamylchloridtreated trypsin preparation used.

The isolation of [14C]Ac‐Ser‐Gln‐Gly both after degradation of the tryptic N‐[14C]acetyl‐peptides by carboxypeptidase A and tryptic cleavage of the peptic O‐[14C]acetyl‐nonapeptide clearly demonstrated, that these peptides are derived from a common parent sequence in the acetyl transferase enzyme.

The above observations revealed the partial sequence of the active centre of the acetyl transferase to be‐Ala‐(Gly2,His,Thr)‐Lys‐Ser([14C]Ac)‐Gln‐Gly‐Leu‐Val‐Thr‐Ala‐Val‐.

By comparing the amino acid composition of [14C]malonyl‐peptides, stable towards performic acid, with the partial sequence of the [14C]acetyl‐enzyme it was concluded that acetyl and malonyl transfer reactions are catalyzed by enzyme components of the multienzyme complex, which are distinctly different. In both cases however, the catalytic mechanisms probably involve the participation of serine residues as acyl carrier groups.