2-oxo Acid Dehydrogenase Complex Research Articles

Rapid purification of bovine kidney branched-chain 2-oxoacid dehydrogenase complex containing endogenous kinase activity

Branched-chain 2-oxoacid dehydrogenase complex has been purified to near homogeneity by a simple, rapid procedure. The final product contains endogenous kinase activity capable of phosphorylating and inactivating the complex. Phosphorylation continues after complete inactivation, indicating the possibility of several phosphorylatable sites.

Mobility and active-site coupling in 2-oxo acid dehydrogenase complexes.

The 2-oxo acid dehydrogenase complexes consist of multiple copies of each of three enzymes, 2-oxo acid decarboxylase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3), which catalyse successive steps in the overall reaction. The complexes are based on a structural core made up of the E2 chains, which also contain lipoic acid residues covalently attached to lysine residues. These lipoic acid residues are involved in transferring the substrate between the different active sites. A combination of limited proteolysis and 1H NMR experiments has shown that the E2 component has an unusual structure, having a substantial segment of polypeptide chain in the form of a highly flexible random coil. This flexibility allows the lipoyl-lysine residues to move rapidly over considerable distances, and provides a mechanism for the system of active-site coupling observed in these complexes.

Activation of phosphorylated branched chain 2-oxoacid dehydrogenase complex

The branched chain 2-oxoacid dehydrogenase complexes of ox kidney [ 11, rat heart, kidney and liver [2-61 and rabbit liver [7] are inactivated by phosphorylation with ATPMg and some aspects of the regulation of the kinase reaction have been described [S]. Little is known of reactivation of phosphorylated complex, although its occurrence in mitochondria is documented 12-41. We show here that phosphorylated ox kidney BCDH complex is activated without dephosphorylation by a high speed supernatant fraction prepared from extracts of rat liver or kidney mitochondria. Activator was not detected in a similar fraction of rat heart mitochondria. Activator was thermolabile, non-diffusible by dialysis, inactivated by trypsin and precipitated by (NI&hS04. complex were omitted, in order to increase the yield of BCDH complex (S-IO-fold) and the rate of inactivation by MgATP ( -4-fold). Apparent first order rate constants for inactivation by MgATP ranged from l-3. Activity of pyruvate dehydrogenase complex was up to 80% of that of BCDH complex as estimated by the activity ratio with ketoleucine and pyruvate [I]. Pig heart pyruvate dehydrogenase complex was purified as in [9].

Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity.

A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 46000-mol.wt. subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labelled enzyme. These results demonstrate that BCOAD can be regulated by an endogenous protein kinase and that the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.

Inactivation of purified ox kidney branched-chain 2-oxoacid dehydrogenase complex by phosphorylation

Inactivation of purified ox kidney branched-chain 2-oxoacid dehydrogenase complex by phosphorylation

Conformational mobility of polypeptide chains in the 2-oxo acid dehydrogenase complexes from ox heart revealed by proton NMR spectroscopy

Conformational mobility of polypeptide chains in the 2-oxo acid dehydrogenase complexes from ox heart revealed by proton NMR spectroscopy

Inactivation of rat liver and kidney branched chain 2-oxoacid dehydrogenase complex by adenosine triphosphate

Inactivation of rat liver and kidney branched chain 2-oxoacid dehydrogenase complex by adenosine triphosphate

Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.

A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.

Active and inactive forms of branched-chain 2-oxoacid dehydrogenase complex in rat heart and skeletal muscle

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4-Methyl-2-oxopentanoate oxidation by rat skeletal-muscle mitochondria.

1. Oxidative decarboxylation of 4-methyl-2-oxopentanoate (2-oxoisocaproate) by mitochondria of rat skeletal muscle showed biphasic kinetics. Two apparent Km values of 9.1 micronM and 0.78 mM were established. In broken mitochondria the rate of oxidation was lower and only the higher apparent Km value was found. 2. Isovalerylcarnitine inhibited 4-methyl-2-oxopentanoate oxidation in the presence and absence of carnitine, but isovaleryl-CoA had no inhibitory effect. 3. Addition of ADP enhanced 4-methyl-2-oxopentanolate oxidation. Malate, succinate and 2-oxoglutarate additionally increased the rate of oxidation, but in the absence of ADP succinate and 2-oxoglutarate inhibited. 4. Addition of rotenone and simultaneous addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and valinomycin markedly decreased 4-methyl-2-oxopentanoate oxidation. 5. These observations indicate that the branched-chain 2-oxo acid dehydrogenase complex is situated on the inner side of the mitochondrial inner membrane. 6. In mitochondria and homogenates CO2 was only produced by oxidative decarbosylation of 4-methyl-2-oxopentanoate. In intact muscle oxidation of this oxo acid proceeds more to completeness. 7. The physiological significance of intermediate formation during oxidation of branched-chain amino acids is discussed.

Inactivation of rat heart branched-chain 2-oxoacid dehydrogenase complex by adenosine triphosphate

Inactivation of rat heart branched-chain 2-oxoacid dehydrogenase complex by adenosine triphosphate

Selective inactivation of the transacylase components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli

1. The reaction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with maleimides was examined. In the absence of substrates, the complex showed little or no reaction with N-ethylmaleimide. However, in the presence of pyruvate and N-ethylmaleimide, inhibition of the pyruvate dehydrogenase complex was rapid. Modification of the enzyme was restricted to the transacetylase component and the inactivation was proportional to the extent of modification. The lipoamide dehydrogenase activity of the complex was unaffected by the treatment. The simplest explanation is that the lipoyl groups on the transacetylase are reductively acetylated by following the initial stages of the normal catalytic cycle, but are thereby made susceptible to modification. Attempts to characterize the reaction product strongly support this conclusion. 2. Similarly, in the presence of N-ethylmaleimide and NADH, much of the pyruvate dehydrogenase activity was lost within seconds, whereas the lipoamide dehydrogenase activity of the complex disappeared more slowly: the initial site of the reaction with the complex was found to be in the lipoyl transacetylase component. The simplest interpretation of these experiments is that NADH reduces the covalently bound lipoyl groups on the transacetylase by means of the associated lipoamide dehydrogenase component, thereby rendering them susceptible to modification. However, the dependence of the rate and extent of inactivation on NADH concentration was complex and it proved impossible to inhibit the pyruvate dehydrogenase activity completely without unacceptable modification of the other component enzymes. 3. The catalytic reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by NADH in the presence of the pyruvate dehydrogenase complex was demonstrated. A new mechanism for this reaction is proposed in which NADH causes reduction of the enzyme-bound lipoic acid by means of the associated lipoamide dehydrogenase component and the dihydrolipoamide is then oxidized back to the disulphide form by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). 4. A maleimide with a relatively bulky N-substituent, N-(4-diemthylamino-3,5-dinitrophenyl)maleimide, was an effective replacement for N-ethylmaleimide in these reactions with the pyruvate dehydrogenase complex. 5. The 2-oxoglutarate dehydrogenase complex of E. coli behaved very similarly to the pyruvate dehydrogenase complex, in accord with the generally accepted mechanisms of the two enzymes. 6. The treatment of the 2-oxo acid dehydrogenase complexes with maleimides in the presence of the appropriate 2-oxo acid substrate provides a simple method for selectively inhibiting the transacylase components and for introducing reporter groups on to the lipoyl groups covalently bound to those components.

Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.