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

ABSTRACT: 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.

SUBMITTER: Brown JP

PROVIDER: S-EPMC1172848 | biostudies-other | 1976 May

REPOSITORIES: biostudies-other

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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).

Project description:Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess 'moonlighting' activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions. Various known affectors of physiological activity and ROS production, including Ca(2+), ADP, lipoylation status or pH, were investigated. The human complexes were also reconstituted with the most prevalent human pathological mutant of the E3 component, G194C and characterized; isolated human E3 with the G194C substitution was previously reported to have an enhanced ROS generating capacity. It is demonstrated that: i. PDHc, similarly to OGDHc, is able to generate ROS and this feature is displayed by both the E. coli and human complexes, ii. Reconstituted hPDHc generates ROS at a significantly higher rate as compared to hOGDHc in both the forward and the reverse reactions when ROS generation is calculated for unit mass of their common E3 component, iii. The E1 component or E1-E2 subcomplex generates significant amount of ROS only in hOGDHc; iv. Incorporation of the G194C variant of hE3, the result of a disease-causing mutation, into reconstituted hOGDHc and hPDHc indeed leads to a decreased activity of both complexes and higher ROS generation by only hOGDHc and only in its reverse reaction.

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

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