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, radioactive malonyl and methylmalonyl enzymes are formed and may be precipitated with trichloroacetic 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.Methy1-[3-W]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methy1)malonyl peptides were separated into three Werent fractions A, B and C. I n peptides B and C (methy1)malonic acid is bound by performic acid stable linkages, whereas (methy1)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor satellite 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]malonyI peptide B was further purified by the following procedures : Dowex-BOX2 chromatography, subtilisin S digestion, DEAE-Sephadex chromatography, Dowex-50 X 2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B, end B,, resulted from subtilisin S treatment. They h l l y were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: Bl = Leu,Ala,Gly,Ser,His and B, = Leu, Ah,Gly(S x),Glu,Ser,Hk. Leucine was the C-terminal amino acid in peptide Bl. No cysteine was found in either peptide.A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAESephadex, Dowex-50 X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl peptide B,.The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methy1)malonic acid in peptides B and C is bound to serine by an 0-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, 8-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'-phosphopantetheine. 4'-Phospho-pantetheine, therefore, represents the so called "central" SH-group in yeast fatty acid synthetase. The other malonyl binding site is thought to be the active oenter of the malonyl transferase in the complex.After alkali treatment of the purified fatty acid synthetase under defined conditions 4'-phosphopantetheine was isolated from the hydrolysate and characterized as S-benzoyl-4'-phosphopantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules ...
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