Catalytic and structural properties of the dihydrolipoyl transacylase component of bovine branched-chain α-keto acid dehydrogenase

D. T. Chuang, C. W C Hu, L. S. Ku

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Abstract

Branched-chain α-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain α-keto acid decarboxylase (E1), dihydrolipoyl transcyclase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain α-keto acid dehydrogenase from bovine kidney and liver was characterized with an independent radiochemical assay for this component. The assay uses the model reaction: R-14CO-S-CoA + Lip-(SH)2 ⇆ R-14CO-S-Lip-SH + CoA-SH, which is similar to that catalyzed by the transacetylase component of the pyruvate dehydrogenase complex. In this reaction, exogenous dihydrolipoamide substitutes for the protein (E2)-bound dihydrolipoyl moiety, and [1-14C]acyl-CoA synthesized enzymatically is the acyl-CoA substrate. The thioester structure of the reaction product, S-acyldihydrolipoamide, was identified by mass spectrometry, its characteristic absorption at 232-245 nm and by formation of hydroxamate with hydroxylamine. Rates of the E2-catalyzed transacylation reaction with various [1-14C]acyl-CoAs are in the order of [1-14C]isobutyryl-CoA > [1-14C]isovaleryl-CoA > [1-14]Cacetyl-CoA. The activity with acetyl-CoA is 15% of that with isobutyryl-CoA. The E2 activity is strongly inhibited by arsenite. Modification of the covalently bound lipoyl moiety through reductive acylation in the presence of N-ethylmaleimide is without effect on the transacylation reaction. These data, along with results of initial velocity and product inhibition suggest that the model reaction proceeds via a random Bi Bi mechanism. Limited proteolysis of purified bovine liver branched-chain α-keto acid dehydrogenase with trypsin results in complete loss of the overall activity catalyzed by the complex. Nonetheless, the activity of the E2 component is not affected. The tryptic digestion cleaves E2 subunits (M(r) = 52,600) into a major fragment of M(r) = 25,700. By contrast, E1α and E1β subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain α-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.

Original languageEnglish (US)
Pages (from-to)9277-9284
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number14
StatePublished - Jan 1 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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