No abstract

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.

Yeast fatty acid synthetase is inactivated by several SH reagents. For the irreversible inhibition by iodoacetamide, the apparent second-order rate constant k = 1.3 f 0.2 M-' sC1 was found (0 "C, pH 6.5). This value is about 125-times higher than the rate constant for the reaction between iodoacetamide and free cysteine under the same conditions and was found to be pH-independent in the range between pH 5 and pH 9. A possible explanation for this result might be that the SH-group alkylated by iodoacetamide is hydrogen-bonded to a neighbouring basic group in the protein.Of all partial activities of the synthetase, only the condensation reaction is impaired. The enzyme can be protected against iodoacetamide by prior treatment with acetyl-CoA but not malonyl-CoA. This indicates that iodoacetamide reacts with peripheral

When yeast fatty acid synthetase is treated with iodoacetamide at 0°C, three carbamoylmethyl residues are incorporated into the protein under concomitant complete loss of the enzymatic activity [Oesterhelt, D., Bauer, H., Kresze, G.‐B., Steber, L. and Lynen, F. (1977) Eur. J. Biochem. 79, 173–180]. Here we report that, in this reaction, only cysteinyl residues are alkylated. During protein hydrolysis of the modified enzyme, S‐carboxymethyl cysteine is formed which, when treated with performic acid in the presence of a strong acid and chloride ions, is oxidized only partially to yield open‐chain S‐carboxymethyl cysteine sulfone but, for the greater part, is transformed into S‐carboxymethyl cysteine lactam sulfone (3‐carboxy‐5‐oxo‐tetrahydro‐1,4‐thiazine‐1,1‐dioxide), a novel derivative of S‐carboxymethyl cysteine which cannot be detected with ninhydrin. If, on the other hand, the carbamoylmethylated enzyme is treated first with performic acid and hydrolyzed afterwards, a large part of the carbamoylmethyl residues is lost due to decarboxylation of the α‐sulfonyl carboxylic acid S‐carboxymethyl cysteine sulfone. When carboxamidomethylated fatty acid synthetase was digested with trypsin at 0°C, two different carbamoylmethyl peptides were found which could be separated by gel filtration. The larger peptide TA was cleaved, during prolongated incubation with trypsin at 30°C, to yield the smaller peptide TB which was purified and its amino acid sequence determined to be Thr‐Pro‐Val‐Gly‐Ala‐Cys(carbamoylmethyl). By comparison of tryptic and peptic peptides from acetylated and carboxamidomethylated synthetase, respectively, it is demonstrated that the same cysteinyl residues are alkylated by iodoacetamide which are the acetyl‐binding sites of the condensing enzyme component, i.e. the peripheral SH‐groups.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.