Search

Malonic acid as well as its model substrate methylmalonic acid are covalently bound to two chemically different sites of the fatty acid synthetase complex of yeast. When radioactively labeled malonyl CoA or methylmalonyl CoA are incubated with the multienzyme complex, radio- active malonyl and methyhnalonyl enzymes are formed and may be precipitated with trichloro- acetic acid. Treatment with performic acid splits only a minor proportion of the (methyl) malonyl enzyme bonds. Most of them are resistant to the oxidant and, hence, prove to be non-thioester linkages. Methyl- [3-14C]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methyl)malonyl peptides. were separated into three different fractions A, B and C. In peptides B and C (methyl)malonic acid is bound by performic acid stable linkages, whereas (methyl)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor sateffite of peptide B, the quantitative relation of both varying somewhat in different preparations. it is concluded that both are derived from a common original peptide sequence in the enzyme. Methyl- [3-14C]malonyl peptide B was further purified by the following procedures: Dowex-50X2 chromatography, subtilisin S digestion, DEAB-Sephadex chromatography, Dowex-50X2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B1and B2, resulted from subtilisin S treatment. They finally were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: B1 = Leu,AIa,Gly,Ser,His and B2 = Leu, Ala,Gly(2 × ),Glu,Ser,His, Leucine was the C-terminal amino acid in peptide B1. No cysteine was found in either peptide. A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAE- Sephadex, Dowex-50X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl B1peptide B2. The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methyl)malonic acid in peptides B and C is bound to serine by an O-ester linkage which is unusually activated by some surrounding amino acid, possibly histidine, [3-14C]Malonal peptide A was shown to contain stoichiometric amounts of cysteamine, β-alanine, and organic phosphate, corresponding to the quantity of malonate bound. These data suggest a thioester linkage between malonate and the SH-group of enzyme-bound 4'-phospho- pantetheine. 4'- Phospho-pantetheine, therefore, represents the so called "central" SR-group in yeast fatty acid synthetase, The other malonyl binding site is thought to be the active center of the malonyl transferase in the complex. After alkali treatment of the purified fatty acid synthetase under defined conditions 4' -phospho - pantetheine was isolated from the bydrolysate and characterized as S-benzoyl-4'-phospho- pantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules of 4'-phospho-pantetheine. According to the amount of malonyl enzyme formed, not all of the enzyme-bound phospho-pantetheine residues accept malonic acid. Some may be involved in other functions. [ABSTRACT FROM AUTHOR]

Presents an investigation of the sequencing of 65 amino acid residues at the carboxyl-terminus. Use of methods such as enzymic or chemical cleavages of the peptide chain; Fractionation of peptides by Sephadex or Dowex column chromatography, paper elctrophoresis or chromatography.