Abstract
Branched-chain alpha-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain alpha-keto acid decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain alpha-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 in equilibrium 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 greater than [1-14C] isovaleryl-CoA greater than [1-14C]acetyl-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 alpha-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 (Mr = 52,600) into a major fragment of Mr = 25,700. By contrast, E1 alpha and E1 beta subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.
Highlights
The thioester structure with the pyruvate dehydrogenase complex [1].The El and E2 of the reaction product, 5’-acyldihydrolipoamide, was componentsareapparently specific for branched-chain aidentified by mass spectrometry,its characteristic ab- keto acid dehydrogenase, while the EBcomponent is common sopriton a t 232-245 nm and by formation of hydrox- to branched-chain a-keto acid, pyruvate, and a-ketoglutarate amate with hydroxylamine
S-Acetyldihydrolipoamide was enzymatically synthesized and characterized by the same procedures except that acetyl-CoA substituted for acyl-CoA, and that partially purified pyruvate dehydrogenase complex was the enzyme used for the synthesis
The present study indicates that highly purified branchedchain a-keto acid dehydrogenase from bovine kidneyand liver
Summary
Vol 259, No 14, Issue of July 25, ppP. r9i2n7te7d-92in84U,1.S98.A4. Catalytic and Structural Propertieosf the Dihydrolipoyl Transacylase Component of Bovine Branched-chain a-KetoAcid Dehydrogenase*. Limitedproteolysis of component is apparently only loosely associated with the purified bovine liver branched-chain a-keto acid de- complex [8].Recently the subunit structure of E2components hydrogenase with trypsin results in complete loss of of pyruvate and a-ketoglutarate dehydrogenase complex has the overall activity catalyzed by the complex. The column was washed with 2 bovine branched-chain a-keto acid dehydrogenase preparations provide evidence for a transacylation reaction between acyl-CoA and dihydrolipoamide that is catalyzed bya specific. S-Acetyldihydrolipoamide was enzymatically synthesized and characterized by the same procedures except that acetyl-CoA substituted for acyl-CoA, and that partially purified pyruvate dehydrogenase complex (recovered at the polyethylene glyco step of branched-chain cY-keto acid dehydrogenase purification) was the enzyme used for the synthesis. Limited Proteolysis with Trypsin-Highly purified bovine liver branched-chain a-keto acid dehydrogenase (50 pg) was incubated with trypsin (0.5 rg)
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