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Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed `inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex. By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetykransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase. [ABSTRACT FROM AUTHOR]

Yeast fatty acid synthetase possesses very low malonyl-CoA decarboxyIase 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. [ABSTRACT FROM AUTHOR]

When yeast fatty acid synthetase is treated with iodoacetamide at 0 °C, three carbamoyimethyl 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. [ABSTRACT FROM AUTHOR]

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 ± 0.2 M-1 s-1 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 SH-groups of the multienzyme complex. N-Ethylmaleimide, in contrast, inhibits the synthetase in a strongly pH-dependent manner. These kinetic data support the conclusion drawn from earlier studies on the malonyl binding sites of the enzyme [Schweizer, E., Piccinini, F., Duba, C., Günther, S., Ritter, E. and Lynen, F. (1970) Eur. J. Biochem. 15, 483-499] that the reaction of N-ethylmaleimide with another SH-group of the enzyme is responsible for the inactivation. During the reaction with iodoacetamide, fatty acid synthetase activity is completely destroyed after incorporation of about three carbamoylmethyl residues per molecule of enzyme complex. At 0 °C, further alkylation by iodoacetamide could not be detected. Since yeast fatty acid synthetase is proposed to be an A6B6 hexamer, the possibility of half-site reactivity is considered. [ABSTRACT FROM AUTHOR]