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

ABSTRACT: 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.

SUBMITTER: Stanley CJ

PROVIDER: S-EPMC1162192 | biostudies-other | 1980 Oct

REPOSITORIES: biostudies-other

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Project description: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.

Project description:The Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) comprises multiple copies of three enzymes-E1o, E2o, and E3-and transthioesterification takes place within the catalytic domain of E2o. The succinyl group from the thiol ester of S8-succinyldihydrolipoyl-E2o is transferred to the thiol group of coenzyme A (CoA), forming the all-important succinyl-CoA. Here, we report mechanistic studies of enzymatic transthioesterification on OGDHc. Evidence is provided for the importance of His375 and Asp374 in E2o for the succinyl transfer reaction. The magnitude of the rate acceleration provided by these residues (54-fold from each with alanine substitution) suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid-base catalysis. Further evidence ruling out a role in acid-base catalysis is provided by site-saturation mutagenesis studies at His375 (His375Trp substitution with little penalty) and substitutions to other potential hydrogen bond participants at Asp374. Taking into account that the rate constant for reductive succinylation of the E2o lipoyl domain (LDo) by E1o and 2-oxoglutarate (99 s-1) was approximately twofold larger than the rate constant for kcat of 48 s-1 for the overall reaction (NADH production), it could be concluded that succinyl transfer to CoA and release of succinyl-CoA, rather than reductive succinylation, is the rate-limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2-oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl-CoA formation, so important for several biological processes including post-translational succinylation of protein lysines.2-oxoglutarate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html); dihydrolipoamide succinyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html); dihydrolipoamide dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html); pyruvate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html); dihydrolipoamide acetyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html).

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

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