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

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.