The palmityl binding sites of fatty acid synthetase from yeast.

Fatty acid synthetase was covalently labelled with [14C]palmitic acid from [14C]palmityl-CoA. Tryptic and peptic digestion of the [14C]palmityl enzyme resulted in the formation of radioactive palmityl peptides carrying the long-chain acyl residue both in oxygen-ester and thio-ester linkage. The lipophilic palmityl peptides were purified by column and thin-layer chromatography using organic lolvent systems. Peptides arising from the acyl carrier protein, the condensing enzyme and the palmityl transferase were identified and characterized. The amino acid sequence of a 4'-phosphopant-etheine-containing peptide was established. It comprises 13 residues and shows a high degree of homology with the acyl carrier protein from Escherichia coli. A heptapeptide and an octapeptide from the palmityl transferase active site were partially sequenced. The identical amino acid composition of palmityl transferase and malonyl transferase core peptides is briefly discussed.     

The isolation of the two subunits of yeast fatty acid synthetase.

The two subunits that comprise the yeast fatty acid synthetase (designated α and β) have been isolated. The separation was performed using DEAE Biogel A chromatography after first treating yeast fatty acid synthetase with 3,4,5,6 tetrahydrophthalic anhydride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fractions eluted from the ion exchange column indicated that the separation of the subunits was essentially complete. It was possible to remove the 3,4,5,6 tetrahydrophthalate derivative from the subunits and regenerate certain of the partial activities. The α subunit was found to have the β-keto reductase activity as well as the acyl carrier protein component associated with it. The β subunit had the acetyl and malonyl transacylases and the palmitoyl transferase activity associated with it. The different extent to which the malonyl and acetyl transacylase activities were regained indicated that these two catalytic sites have separate domains in the β subunit.     

Origin of hydrogen atoms in the fatty acids synthesized with yeast fatty acid synthetase.

The mechanism of hydrogen incorporation into fatty acids was investigated with an enzyme preparation from baker's yeast. Fatty acids synthesized from malonyl-CoA and acetyl-CoA in the presence of D2O or stereospecifically deuterium-labeled NADPH were isolated and analyzed by mass chromatography to examine the localization of deuterium atoms in the molecule. The following results were obtained: 1. Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom). The second hydrogen atom was incorporated as the result of hydrogen exchange phenomenon between the methylene group of malonyl CoA and water. 2. HB hydrogen of NADPH was used for beta-ketoacyl reductase. 3. HB hydrogen of NADPH was also used for enoyl reductase. 4. Hydrogen atoms from HB position of NADPH were found on the odd-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom).     

Simple assay for the condensation component enzyme (beta-ketoacyl synthetase) of fatty acid synthetase.

A simple assay is described for estimating the activity of the condensation component enzyme (beta-ketoacyl synthetase) of the yeast fatty acids synthetase complex. The radioactivity liberated as 14CO2 from [1,3-14C]malonyl-CoA was trapped in phenethylamine and measured by liquid scintillation spectroscopy. Three enzyme-catalysed steps are involved: acetyl-CoA transacylase, malonyl-CoA transacylase and beta-ketoacyl synthetase; however, beta-ketoacyl synthetase is rate-limiting. beta-Ketoacyl synthetase activity was made independent of subsequent enzyme activities of the complex by excluding NADPH from the assay, thus blocking beta-ketoacyl reductase and preventing fatty acid synthesis. By this assay beta-ketoacyl synthetase activity was about 0.28 of the activity of the complex for fatty acid synthesis, compared with approximately 0.001 for published assays. Several pyridine nucleotides and derivatives were tested after it was discovered that NADH stimulated beta-ketoacyl synthetase activity to a greater extent than could be accounted for by its reactivity in providing a pathway from acetoacetyl-enzyme to fatty acid synthesis. Presumably, the release of acetoacetate from the central sulphydryl of the complex is the rate-limiting step in the assay procedure.     

Reaction of yeast fatty acid synthetase with iodoacetamide. 3. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide.

Yeast fatty acid synthetase possesses very low malonyl-CoA decarboxylase activity. Treatment with iodoacetamide, while abolishing synthetase activity, induces a strong malonyl decarboxylase activity which, in turn, can be inhibited by N-ethylmaleimide. Kinetic analysis shows that the emergence of the decarboxylase activity is synchronized to the disappearance of the fatty-acid-synthesizing activity and thus, is due to carboxamidomethylation of the peripheral SH-groups of the multienzyme complex. Strong decarboxylase activity was also found after treatment of the synthetase with methylmalonyl-CoA. A hypothetical scheme is proposed which explains the origination of the decarboxylase activity as a consequence of conformational changes of the condensing enzyme component which happen when the peripheral SH-group is acylated or alkylated.     

tRNA-dependent amino acid discrimination by yeast seryl-tRNA synthetase.

The ability of aminoacyl-tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) employs tRNA-dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., S?ll, D. & Weygand-Durasevic, I. (1997) J. Biol. Chem.272, 1136-1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl-adenylate formation than the apoenzyme alone. Sequence-specific tRNA-protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA-induced conformational change in the enzyme's active site. The 3'-terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA-assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.     

Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.

The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes.