Search PubMed⌕ Search

Biomedical subjects

M J Danson

Publications and source records attributed to M J Danson.

71 records · Page 4Linked to original sources
PubMed1984

Dihydrolipoamide dehydrogenase from halophilic archaebacteria.

Dihydrolipoamide dehydrogenase has been discovered in the halophilic archaebacteria for the first time. The enzyme from both classical and alkaliphilic halobacteria has been investigated. (1) The enzyme specifically catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+. Enzymic activity was optimal at 2 M-NaCl and was remarkably resistant to thermal denaturation. (2) The relative molecular masses (Mr) of the native enzyme from the various species of halobacteria were determined to be within the range 112000-120000. (3) The enzyme exhibited a hyperbolic dependence of catalytic activity on both dihydrolipoamide and NAD+ concentrations. From these steady-state kinetic measurements the dissociation constant (Ks) of dihydrolipoamide was determined to be 57 (+/- 5) microM. (4) The enzyme was only susceptible to inactivation by iodoacetic acid in the presence of its reducing ligands, dihydrolipoamide or NADH. The rate of inactivation followed a hyperbolic dependence on the concentration of dihydrolipoamide, from which the Ks of this substrate was calculated to be 55 (+/- 7) microM. Together with the steady-state kinetic data, the pattern of inactivations is consistent with the involvement in catalysis of a reversibly reducible disulphide bond, as has been found in dihydrolipoamide dehydrogenase from non-archaebacterial species. In eubacterial and eukaryotic organisms, dihydrolipoamide dehydrogenase functions in the 2-oxo acid dehydrogenase complexes. These multienzyme systems have not been detected in the archaebacteria, and, in the context of this apparent absence, the possible function and evolutionary significance of archaebacterial dihydrolipoamide dehydrogenase are discussed.

Archaea↗
PubMed1983

Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme.

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.

Amino Acids↗
PubMed1981

The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component. It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction. There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+. However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity. The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.

Acetyl Coenzyme A↗
PubMed1981

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes from bacterial and mammalian sources.

A simple method was developed for assessing the intramolecular coupling of active sites in the lipoate acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complexes from Escherichia coli, Bacillus stearothermophilus and ox heart and pig heart mitochondria. Samples of enzyme complex were prepared in which the pyruvate decarboxylase (E1) component was selectively and partly inhibited by treatment with increasing amounts of a transition-state analogue, thiamin thio-thiazolone pyrophosphate. The fraction of the E2 component acetylated by incubation with [2-14C] pyruvate, in the absence of CoA, was determined for each sample of partly inhibited enzyme and was found in all cases to exceed the fraction of overall complex activity remaining. This indicated the potential for transacetylation reactions among the lipoic acid residues within the E2 core. A graphic presentation of the data allowed comparison of the active-site coupling in the various enzymes, which may differ in their lipoic acid content (one or two residues per E2 chain). It is clear that active-site coupling is a general property of pyruvate dehydrogenase complexes of octahedral and icosahedral symmetries, the large numbers of subunits in each E2 core enhancing the effect.

Acetylation↗
PubMed1980

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.

Escherichia coli↗
PubMed1979

Studies on a mutant form of Escherichia coli citrate synthase desensitised to allosteric effectors.

Naturally occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a 'large' enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as Escherichia coli, also by 2-oxoglutarate. On the other hand, Gram-positive bacteria and all eukaryotes produce a 'small' citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies we have explored the possibility of genetically altering one type of citrate synthase to the other. By mutagenesis and suitable selection we have succeeded in isolating a mutant of E. coli whose citrate synthase is both 'small' and insensitive to NADH and 2-oxoglutarate. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.

Allosteric Regulation↗
PubMed1978

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.

Binding Sites↗
PubMed1978

Rapid intramolecular coupling of active sites in the pyruvate dehydrogenase complex of Escherichia coli: mechanism for rate enhancement in a multimeric structure.

In the absence of CoA and presence of pyruvate, the lipoic acid residues covalently bound to the lipoate acetyltransferase core component (acetyl-CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12) of the pyruvate dehydrogenase multienzyme complex of Escherichia coli become reductively acetylated. A study of a series of reassembled complexes varying only in their content of pyruvate decarboxylase [pyruvate:lipoate-oxidoreductase (decarboxylating and acceptor-acetylating) EC 1.2.4.1] showed that the initial direct reductive acetylation of lipoic acid residues can be followed by extensive intramolecular transacetylation reaction between lipoic acid residues on neighboring polypeptide chains of the lipoate acetyltransferase core [Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268, 313-316]. Pulsed-quenched-flow measurements of the rates of the acetylation reactions in the various complexes now demonstrate that the intramolecular transacetylation reactions are not rate-determining in the normal reaction mechanism of the enzyme. There is therefore the potential for rapid multiple coupling of active sites in the lipoate acetyltransferase core. The rate constant for the overall complex reaction, measured by stopped-flow fluorimetry, is found to be approximately twice that for the reductive acetylation reaction measured by pulsed-quenched flow. This result could mean that CoA is an allosteric stimulator of the reductive acetylation part of the overall reaction or that there are two active sites on each chain of the lipoate acetyltransferase component working in parallel. A system of rapid functional connection of active sites in a multienzyme complex ensures that sequential reactions can be successfully coupled even under conditions of low substrate concentrations for the different steps. The substantial rate enhancement thus achieved offers a plausible explanation for the unusual complexity of the quaternary structure of the enzyme.

Binding Sites↗
PubMed1977

Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.

The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.

Chemical Phenomena↗
PubMed1976

Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The reaction of two maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of Escherichia coli in the presence of the substrate, pyruvate, was examined. In both cases, the reaction was demonstrated to be almost exclusively with the lipoate acetyltransferase component, and evidence is presented to show that the most likely sites of reaction are the lipoic acid residues covalently bound to this component. With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears two functionally active lipolic acid residues. This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.

Acetyltransferases↗
PubMed1976

Citrate synthase.

Explore the source record for details and available documents.

Adenosine Diphosphate↗
PubMed1973

Functional groups in the activity and regulation of Escherichia coli citrate synthase.

1. Citrate synthase has been purified from Escherichia coli and shown to exist at an equilibrium between three forms: monomer (mol.wt. 57000), tetramer (mol.wt. 230000) and, possibly, octamer. Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and alpha-oxoglutarate. 2. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependence of their rates of photo-oxidation with Methylene Blue suggests that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal and the destruction of activity by diethyl pyrocarbonate. 3. The participation of histidine at the alpha-oxoglutarate effector site is indicated by photo-oxidation and the participation of cysteine at the NADH effector site suggested by photo-oxidation is confirmed by the desensitization to NADH produced by treatment with 5,5'-dithiobis-(2-nitrobenzoate). Inactivation of the enzyme after modification with this reagent suggests the additional involvement of cysteine in catalytic activity. 4. Amino acid analyses of native and photo-oxidized enzyme are consistent with these conclusions. 5. Modification with 2-hydroxy-5-nitrobenzyl bromide indicates the participation of tryptophan in the activity of the enzyme.

Amino Acids↗