Publications

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

  Chuang D.T., Hu C.C., Ku L.S., Niu W.L., Myers D.E., Cox R.P.

  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 ... >> More

  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. << Less

  J Biol Chem 259:9277-9284(1984) [PubMed] [EuropePMC]

• Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions.

  Perham R.N.

  Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a num ... >> More

  Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a number of the multifunctional enzyme systems responsible. The protein domains, for which the posttranslational machinery in the cell is highly specific, are crucially important, contributing to the processes of molecular recognition that define and protect the substrates and the catalytic intermediates. The domains have novel folds and move by virtue of conformationally flexible linker regions that tether them to other components of their respective multienzyme complexes. Structural and mechanistic imperatives are becoming apparent as the assembly pathways and the coupling of multistep reactions catalyzed by these dauntingly complex molecular machines are unraveled. << Less

  Annu Rev Biochem 69:961-1004(2000) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Branched-chain amino acid catabolism in bacteria.

  Massey L.K., Sokatch J.R., Conrad R.S.

  Bacteriol Rev 40:42-54(1976) [PubMed] [EuropePMC]