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At least 289 records · Page 16Linked to original sources
PubMed1983

Evidence for a multiple random coupling mechanism in the alpha-ketoglutarate dehydrogenase multienzyme complex of Escherichia coli: a computer model analysis.

A computer modeling system was used to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex accompanying release of lipoic acid residues by lipoamidase and by trypsin [Stepp, L. R., Bleile, D. M., McRorie, D. K., Pettit, F. H. & Reed, L. J. (1981) Biochemistry 20, 4555-4560]. The results provide insight into the active-site coupling mechanism in the alpha-ketoglutarate dehydrogenase complex. The model studies indicate that the overall activity of the alpha-ketoglutarate dehydrogenase complex is influenced by redundancies and random processes that we describe as a multiple random coupling mechanism. More than one lipoyl moiety services each E1 subunit (alpha-ketoglutarate dehydrogenase, EC 1.2.4.2), and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data was obtained with a model that has two lipoyl moieties servicing each E1 subunit and a lipoyl-lipoyl interaction network that links all lipoyl moieties on the E2 cube (dihydrolipoamide succinyltransferase, EC 2.3.1.61). The single lipoyl moiety on an E2 subunit is assumed to service the coenzyme A-dependent succinyltransferase site of that E2 subunit as well as an E3 subunit (dihydrolipoamide dehydrogenase, EC 1.6.4.3) if the latter is bound to that particular E2 subunit.

Acyltransferases↗
PubMed1998

Coordinate transcription and physical linkage of domains in surfactin synthetase are not essential for proper assembly and activity of the multienzyme complex.

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.

Bacillus subtilis↗
PubMed2000

Characterization of the sialidase molecular defects in sialidosis patients suggests the structural organization of the lysosomal multienzyme complex.

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G-->A (Gly227Arg), C893C-->T (Ala298Val), G203G-->T (Gly68Val), A544A-->G (Ser182Gly) C808C-->T (Leu270Phe) and G982G-->A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.

Adolescent↗
PubMed1986

Lipoic acid is the site of substrate-dependent acetylation of component X in ox heart pyruvate dehydrogenase multienzyme complex.

The recently characterized Mr-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl-CoA resulted in a time-dependent diminution of incorporation of radiolabelled N-ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N-ethylmaleimide.

Acetylation↗
PubMed1992

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗
PubMed1997

Information transfer in multienzyme complexes--2. The role of Arg64 of Chlamydomonas reinhardtii phosphoribulokinase in the information transfer between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

A mutant phosphoribulokinase has been isolated from the 12-2B mutant of Chlamydomonas reinhardtii. In this mutant, Arg64 has been replaced by Cys. The enzyme, which may exist in the dimeric and tetrameric states, is almost devoid of activity. Neither of these enzymes is able to form a complex with glyceraldehyde-3-phosphate dehydrogenase. The phosphoribulokinase gene has been expressed in Escherichia coli. The resulting recombinant protein, after isolation and purification, is apparently identical to the native enzyme extracted from the chloroplast. Three mutants have been generated by site directed mutagenesis. Arg64 has been replaced by Ala, Lys or Glu. With the exception of the latter, the two other mutants, [A64]phosphoribulokinase and [K64]phosphoribulokinase, are active when they are reduced, and nearly totally inactive in their oxidized state. Their activity, however, is decreased relative to that of the native, or to that of the wild-type recombinant phosphoribulokinase. Both the catalytic constant and the apparent affinity of ribulose 5-phosphate are decreased relative to the corresponding values obtained for the wild-type, the native or the recombinant enzyme. Whereas the [A64]phosphoribulokinase is unable to form a complex with glyceraldehyde-3-phosphate dehydrogenase, [K64]phosphoribulokinase does, but the stability of the resulting complex is much decreased relative to that of the wild-type complex. The oxidized mutant [K64]phosphoribulokinase becomes active in the presence of glyceraldehyde-3-phosphate dehydrogenase but this activity is smaller than that of the corresponding wild-type enzyme. Taken together, these results show that Arg64 plays a major role in the association of the two enzymes and in the information transfer that takes place between glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. As this residue also appears to be important for catalytic activity, it may be tempting to consider that it stabilizes a conformation that is required for both the catalytic activity and the formation of the bienzyme complex.

Animals↗
PubMed1985

Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.

Aconitic Acid↗
PubMed1988

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium.

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.

Amino Acid Sequence↗