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PubMed1986

Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptible to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the--AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH--group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.

Acetylation↗
PubMed1989

Essential role of T4 phage deoxycytidylate hydroxymethylase in a multienzyme complex for deoxyribonucleotide synthesis.

Several enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis interact in T4 phage-infected Escherichia coli to form a multienzyme aggregate, the T4 dNTP synthetase complex. To test the specificity of enzyme interactions seen in vitro with this complex, we analyzed bacteria infected with four T4 gene 42 amber mutants, which specify truncated forms of dCMP hydroxymethylase, one of the constituent enzymes in the complex. Mutants that specify a nearly full length gene 42 product can form an intact complex, as revealed by two criteria: kinetic coupling among constituent enzymes in crude extracts of infected bacteria, and co-elution of enzyme activities from a gel filtration column. By these criteria, mutations that specify truncated proteins less than half the size of the full length protein cause disruption of the complex. These findings suggest that an enzymatically inactive form of dCMP hydroxymethylase can contribute toward assembly of an intact complex, so long as the incomplete protein is of sufficient size to fold normally, allowing interaction with other proteins in the complex.

Chromatography, Gel↗
PubMed1986

Immunological and biosynthetic studies on the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

High-titre, monospecific, polyclonal antisera have been raised against purified mitochondrial 2-oxoglutarate dehydrogenase complex (OGDC) from ox heart and two of its three constituent enzymes, 2-oxoglutarate dehydrogenase (E1) and lipoyl succinyltransferase (E2). These specific antisera have been employed to monitor molecular events in the biosynthesis, import and maturation of this multimeric assembly. Lipoamide dehydrogenase (E3) elicits a poor antibody response in comparison to the other polypeptides of the complex. In cultured pig kidney cells (PK-15), incubated with [35S]methionine in the presence of uncouplers of oxidative phosphorylation, appearance of stable higher-Mr forms of the individual enzymes can be detected by specific immunoprecipitation and fluorographic analysis. In the case of 2-oxoglutarate dehydrogenase, E1, the initial cytoplasmic translation product has a subunit Mr value of 1500-3000 greater than in the mature enzyme while the precursor of the lipoyl succinyltransferase, E2, contains an additional sequence of Mr 6000-8000. Competition studies have revealed the immunological similarity of the precursor molecules to the native subunits. On removal of uncouplers, processing of accumulated precursors is rapidly initiated and is complete within 40 min. Interestingly, antiserum to native 2-oxoglutarate dehydrogenase complex fails to recognise E2 precursor molecules (pre-E2), which can be immunoprecipitated, however, by antibodies raised against the denatured E2 subunit. It is concluded that pre-E2 is conformationally dissimilar to native E2, which exists normally as a highly ordered, multimolecular aggregate in the native complex.

Animals↗
PubMed1986

Glycine decarboxylase multienzyme complex. Purification and partial characterization from pea leaf mitochondria.

The P, H, and T proteins of the glycine cleavage system have been purified separately from pea leaf mitochondria and demonstrate molecular weights of 98,000, 15,500, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of P protein by gel filtration was 210,000, indicating that this enzyme has a native homodimer conformation. Reconstitution assays containing purified P, H, and T proteins and yeast lipoamide dehydrogenase catalyze the oxidation of glycine and demonstrate a strict dependence on pyridoxal phosphate, tetrahydrofolate, NAD+, and dithiothreitol. The released CO2, methylamine-H protein intermediate, and methylenetetrahydrofolate are produced in stoichiometric amounts from glycine during the cleavage reaction. H protein acts as co-substrate with glycine during the decarboxylation reaction, demonstrating an apparent Km value of 2.2 microM. P and H protein alone jointly catalyze the glycine carboxyl-14 CO2 exchange reaction in the presence of pyridoxal phosphate and dithiothreitol. L protein of the glycine cleavage system was immunopurified using monoclonal antibodies. Antigenic and molecular weight similarities of the L protein with the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex were shown suggesting the possibility of common isomers of lipoamide dehydrogenase for the two enzyme complexes.

Amino Acid Oxidoreductases↗
PubMedDate not supplied

[Symmetry of multienzyme complexes].

A model for studying the symmetry of stable states arising from polyenzymic complex conformations is proposed. A formal scheme of submolecular structure self-assembly, on which the model is based, enables it not only to limit the class of conformations but in some cases to determine the structure of a complex in an unambigous manner. The model is shown in its application to polyenzymic complexes of dehydrogenases of alpha-keto acids.

Azotobacter↗