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PubMed1976

Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart.

The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.

Animals↗
PubMed1991

On the FAD-induced dimerization of apo-lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens. Kinetics of reconstitution.

The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197-207]. The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochim. Biophys. Acta 159, 244-256]. The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of starting material. Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two-step sequential process. Initial flavin-binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum. The kinetics of FAD-induced dimerization are strongly dependent on the apoenzyme used. At 0 degrees C, the monomeric apoenzyme-FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH.

Animals↗
PubMed1989

Deficiency of pyruvate dehydrogenase complex (PDHC) in Leigh's disease fibroblasts: an abnormality in lipoamide dehydrogenase affecting PDHC activation.

Several groups have reported abnormalities of the pyruvate dehydrogenase complex (PDHC) in cultured cells or other tissues from patients with Leigh's disease (subacute necrotizing encephalomyelopathy). We therefore undertook studies to elucidate the molecular basis of the defect of PDHC in cultured skin fibroblasts from two patients with Leigh's disease. The deficit of total PDHC activity in homogenates of Leigh's disease fibroblasts could be restored by adding exogenous lipoamide dehydrogenase (LAD, E3), the third component of PDHC. The LAD in these Leigh's disease cells had a markedly reduced ability (less than 20% of normal LAD) to reconstitute with other PDHC components to form active enzyme complex. A polyclonal antibody to pig heart LAD inhibited LAD activity in control cells more efficiently than in Leigh's disease cells. Other mitochondrial enzyme activities and growth of these two Leigh's disease cells appeared normal. These results suggest that the deficiency of PDHC in these two patients with Leigh's disease was due to a structural abnormality of the LAD component of PDHC.

Brain Diseases, Metabolic↗
PubMed1995

Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S.

Lipoamide dehydrogenase from Escherichia coli, a dimeric flavoprotein in the pyridine nucleotide-disulfide oxidoreductase family of enzymes, catalyzes the reduction of NAD+ by dihydrolipoamide. The two electrons are transferred via a redox active disulfide and FAD. Cys44 and Cys49 comprise the redox active disulfide, Cys44 interchanging with dihydrolipoamide and Cys49 interacting with the flavin. Each of these residues has been mutated to serine (C44S, C49S). The altered enzymes showed minute amounts of activity, 0.003% for C44S and 0.012% for C49S using the physiological substrates dihydrolipoamide and NAD+. These very low activities were expected, since the disulfide was no longer present in C44S and C49S, making dithiol-disulfide interchange impossible. However, the enzymes were capable of catalyzing reactions using NADH as the electron donor and alternate electron acceptors: K3Fe(CN)6, thio-NAD+, DCIP, and O2. These activities with NADH indicated that interaction of C44S and C49S with pyridine nucleotides was not affected greatly by the mutation. The pH dependence of the charge-transfer absorbance of C44S gives pKa values of 2.7, associated with titration of Cys49, and 9.5, associated with titration of the acid-base catalyst, His444'. A pKa of 5.1 was estimated for Cys44 in C49S from the pH dependence of its reactivity with methyl methanethiosulfonate. The fluorescence of the FAD in oxidized wild type lipoamide dehydrogenase is markedly temperature dependent, while the remaining fluorescence of two-electron-reduced enzyme is independent of temperature. The fluorescence of the FAD in C44S and in C49S is likewise independent of temperature. The FAD of C44S and C49S is stoichiometrically titrated by 1 equiv of sodium dithionite. However, the FAD of C44S is markedly less completely reduced by 1 equiv of NADH than is the FAD of C49S. Ferricyanide stoichiometrically reoxidizes the FADH2 of both altered forms of the enzyme.

Cysteine↗
PubMed1995

A lipoamide dehydrogenase from Neisseria meningitidis has a lipoyl domain.

A protein of molecular weight of 64 kDa (p64k) found in the outer membrane of Neisseria meningitidis shows a high degree of homology with both the lipoyl domain of the acetyltransferase and the entire sequence of the lipoamide dehydrogenase, the E2 and E3 components of the dehydrogenase multienzyme complexes, respectively. The alignment of the p64k with lipoyl domains and lipoamide dehydrogenases from different species is presented. The possible implications of this protein in binding protein-dependent transport are discussed. This is the first lipoamide dehydrogenase reported to have a lipoyl domain.

Amino Acid Sequence↗
PubMed2000

Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine decarboxylase multienzyme system. 1. Biochemical studies.

Lipoamide dehydrogenase or dihydrolipoamide dehydrogenase (EC 1.8.1. 4) is the E3-protein component of the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. It is also the L-protein component of the glycine decarboxylase system. Although the enzymology of this enzyme has been studied exhaustively using free lipoamide as substrate, no data are available concerning the kinetic parameters of this enzyme with its physiological substrates, the dihydrolipoyl domain of the E2 component (dihydrolipoyl acyltransferase) of the 2-oxoacid dehydrogenase multienzyme complexes or the dihydrolipoyl H-protein of the mitochondrial glycine decarboxylase. In this paper, we demonstrate that Tris(2-carboxyethyl)phosphine, a specific disulfide reducing agent, allows a continuous reduction of the lipoyl group associated with the H-protein during the course of the reaction catalysed by the L-protein. This provided a valuable new tool with which to study the catalytic properties of the lipoamide dehydrogenase. The L-protein displayed a much higher affinity for the dihydrolipoyl H-protein than for free dihydrolipoamide. The oxidation of the dihydrolipoyl H-protein was not affected by the presence of structurally related analogues (apoH-protein or octanoylated H-protein). In marked contrast, these analogues strongly and competitively inhibited the decarboxylation of the glycine molecule catalysed by the P-protein component of the glycine decarboxylase system. Small unfolded proteolytic fragments of the H-protein, containing the lipoamide moiety, displayed Km values for the L-protein close to that found for the H-protein. On the other hand, these fragments were not able to promote the decarboxylation of the glycine in the presence of the P-protein. New highly hydrophilic lipoate analogues were synthesized. All of them showed Km and kcat/Km values very close to that found for the H-protein. From our results we concluded that no structural interaction is required for the L-protein to catalyse the oxidation of the dihydrolipoyl H-protein. We discuss the possibility that one function of the H-protein is to maintain a high concentration of the hydrophobic lipoate molecules in a nonmicellar state which would be accessible to the catalytic site of the lipoamide dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1989

Titration studies on the active sites of pig heart lipoamide dehydrogenase and yeast glutathione reductase as monitored by the charge transfer absorbance.

Macroscopic pKa values associated with the influence of pH on the visible spectrum of 2-electron reduced pig heart lipoamide dehydrogenase and yeast glutathione reductase have been determined by monitoring changes in the principal flavin band near 460 nm and the charge transfer band at 540 nm. The ionization of at least three active site amino acid side chains can influence the spectra over the range of pH studied: the two nascent thiols (interchange thiol and electron transfer thiol) and the histidine residue which acts as the base catalyst in lipoamide dehydrogenase and the acid catalyst in glutathione reductase thiol-disulfide interchange reactions. These systems are analogous to, but more complex than, those in glyceraldehyde-3-phosphate dehydrogenase and papain where a single thiol and a histidine residue in a relatively apolar milieu form a thiolate-imidazolium ion pair which is favored over the thiol-imidazole prototropic tautomer. In an effort to more nearly mimic the papain titrations, the macroscopic pKa values were determined on reduced glutathione reductase which had been monoalkylated with iodoacetamide under conditions known to favor the reaction of the interchange thiol by at least 10 to 1 (Arscott, L. D., Thorpe, C., and Williams, C. H., Jr. (1981) Biochemistry 20, 1513-1520). Like papain and glyceraldehyde-3-phosphate dehydrogenase, alkylated glutathione reductase showed two macroscopic pKa values, at pH 3.7 and pH 9.1, and by analogy, these were associated primarily with the thiol and the imidazole, respectively. Results with the native enzymes depended on the wavelength monitored. Glutathione reductase had pKa values at 4.8, 7.1, and 9.2 when monitored at 540 nm and 5.1 and 8.2 when monitored at 462 nm. Lipoamide dehydrogenase had pKa values at 4.4 and 8.7 when monitored at 529 nm and 3.9, 7.0, and 9.3 when monitored at 455 nm.

Animals↗
PubMed1982

Optimal conditions for the assay of lipoamide dehydrogenase in homogenized human platelets.

Widely different method have been used to assay lipoamide dehydrogenase in tissues from patients with neurological diseases. We have re-examined conditions of assay in homogenized human platelets in the light of results of optimal and inhibitory conditions others have found for the purified pig and rat liver enzymes. Optimal conditions in homogenized platelets for the forward, physiological direction were pH 8.0, 2-4 mmol/l dihydrolipoamide and 1.6-2 mmol/l NAD+ and for the reverse reaction, pH 7.3, 1.2-2 mmol/l lipoamide and 0.125-0.2 mmol/l NADH. Km values by the Lineweaver-Burke method were approximately 420 mumol/l dihydrolipoamide, 180 mumol/l NAD+, 600 mumol/l lipoamide and 27 mumol/l NADH. The optimal conditions and Km values are similar to those reported for the purified pig and rat enzymes. Assays by the present methods should therefore reflect the activity of lipoamide dehydrogenase and not the effects of substrate or cofactor inhibition nor the effects of other, interfering enzyme activities.

Blood Platelets↗
PubMed1988

X-ray study of baker's yeast lipoamide dehydrogenase at 4.5 A resolution by molecular replacement method.

The molecular structure of lipoamide dehydrogenase from baker's yeast has been determined at 4.5 A resolution by molecular replacement techniques using the known structure of human erythrocyte glutathione reductase as a starting model. The enzyme crystallizes in the space group P2(1)2(1)2(1) with a = 98.6(2), b = 162.0(2), c = 69.4(2) A. There is one molecule per asymmetric unit. The enzyme is a dimeric protein of identical subunits related by a local two-fold symmetry. Comparison of the tertiary structures between glutathione reductase and the present enzyme shows that the folding is almost the same except for the N and C termini, although some slight shortening or shifting of alpha-helices was found in the electron density map. FAD molecules are found at similar positions to those of glutathione reductase. Since the amino acid residues around FAD and NAD binding sites and at the reaction centers of the two enzymes are strongly conserved, the lipoamide dehydrogenase may catalyze the opposite reaction through a similar mechanism to that proposed for glutathione reductase. The newly found C terminus is located near the edge of a deep cave at the interface between the two subunits. These additional 18 residues form a narrow entrance to the cave, in which the long chain of the dihydrolipoyl moiety of lipoate acetyltransferase will be bound.

Crystallization↗
PubMed1991

Superoxide anion production by lipoamide dehydrogenase redox-cycling: effect of enzyme modifiers.

Redox-cycling of porcine heart lipoamide dehydrogenase in the presence of NADH and oxygen produced O2-. (NADH-oxidase activity) as demonstrated by (a) reduction of cytochrome c; (b) reduction of the Fe(III)-ADP complex; (c) lucigenin luminescence and (d) the inhibitory effect of superoxide dismutase. NAD+ and p-chloromercuribenzoate inhibited O2-. generation whereas arsenite enhanced it. Comparison of heart and yeast enzyme preparations revealed a close correlation between lipoamide reductase and NADH-oxidase activities. It is concluded that O2-. production is a molecular property of lipoamide dehydrogenase.

Acridines↗
PubMed1998

Co-regulation of lipoamide dehydrogenase and 2-oxoglutarate dehydrogenase synthesis in Escherichia coli: characterisation of an ArcA binding site in the lpd promoter.

The lipoamide dehydrogenase gene (lpdA) encoding the E3 subunits of both the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes of Escherichia coli, is expressed from the upstream pdh and internal lpd promoters of the pdh operon (pdhR-aceEF-lpdA). Under aerobic conditions, the specific components of the 2-oxoglutarate dehydrogenase complex encoded by the sucAB genes in the sdhCDAB-sucABCD operon are expressed from the sdh promoter. The provision of lipoamide dehydrogenase subunits for assembly into the 2-oxoglutarate dehydrogenase complex could thus be controlled by co-regulation of the lpd promoter with the sdh promoter. Here, the transcription start point of the lpd promoter was defined by primer extension analysis, and an ArcA binding site, TGTTAACAAT, overlapping the lpd promoter and matching the consensus at 8 out of 10 positions, was identified by in vitro footprint analysis. PdhR was not bound to the lpd promoter nor was ArcA bound specifically to the pdh promoter. These results support the view that co-regulation of the lpd and sdh promoters is mediated primarily by ArcA.

Aerobiosis↗
PubMed2001

Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase.

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.

Animals↗
PubMed1992

Pig heart lipoamide dehydrogenase: solvent equilibrium and kinetic isotope effects.

Lipoamide dehydrogenase is a flavoprotein which catalyzes the reversible oxidation of dihydrolipoamide, Lip(SH)2, by NAD+. The ping-pong kinetic mechanism involves stable oxidized and two-electron-reduced forms. We have investigated the rate-limiting nature of proton transfer steps in both the forward and reverse reactions catalyzed by the pig heart enzyme by using a combination of alternate substrates and solvent kinetic isotope effect studies. With NAD+ as the variable substrate, and at a fixed, saturating concentration of either Lip(SH)2 or DTT, inverse solvent kinetic isotope effects of 0.68 +/- 0.05 and 0.71 +/- 0.05, respectively, were observed on V/K. Solvent kinetic isotope effects on V of 0.91 +/- 0.07 and 0.69 +/- 0.02 were determined when Lip(SH)2 or DTT, respectively, was used as reductant. When Lip(SH)2 or DTT was used as the variable substrate, at a fixed concentration of NAD+, solvent kinetic isotope effects of 0.74 +/- 0.06 and 0.51 +/- 0.04, respectively, were observed on V/K for these substrates. Plots of the kinetic parameters versus mole fraction D2O (proton inventories) were linear in all cases. Solvent kinetic isotope effect measurements performed in the reverse direction using NADH as the variable substrate showed equivalent, normal solvent kinetic isotope effects on V/KNADH when oxidized lipoamide, lipoic acid, or DTT were present at fixed, saturating concentrations. Solvent kinetic isotope effects on V were equal to 1.5-2.1. When solvent kinetic isotope effect measurements were performed using the disulfide substrates lipoamide, lipoic acid, or DTT as the variable substrates, normal kinetic isotope effects on V/K of 1.3-1.7 were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli.

Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.

Dihydrolipoamide Dehydrogenase↗
PubMed1977

Immobilised lipoamide dehydrogenase. 2. Properties of the enzyme immobilised to agarose through spacer molecules of various lengths.

1. Pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been immobilised to Sepharose by thiol-disulphide interchange via a series of thiolated spacer molecules of increasing length. A number of properties of the immobilised enzyme have been investigated in order to ascertain the effects of proximity to the matrix backbone. 2. Proximity to the matrix backbone reduced the specific activity for lipoamide as substrate but enhanced by 3-8-fold the diaphorase activity with 2,6-dichloroindophenol. These observations are explained in part by an increase in the apparent Km for lipoamide when the enzyme is covalently attached to Sepharose via a short spacer molecule. 3. Both the thermal stability at 90 degrees C and the stability in 30% (v/v) dioxane are enhanced by up to 200% when the enzyme resides close to the matrix but approach those of the native enzyme as the length of the spacer molecule is increased. 4. These data have been correlated with measures of the accessibility of the enzyme as the nominal length of the spacer arm was increased. Thus, as the chain length increased, the rate of cleavage of the disulphide linkage between the enzyme and spacer increased and the enzyme became more susceptible to proteolysis by thermolysin. In contrast, increasing the chain length of the spacer made the enzyme less amenable to inhibition by a specific antibody. 5. These data are discussed in terms of the effect of the matrix on the conformation of the bound enzyme.

Animals↗
PubMed1981

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a bifunctional arsenoxide: selective inactivation of lipoamide dehydrogenase.

The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli. The ability of the lipoate acetyltransferase (E2) component to bind coenzyme A was decreased by about 50% in this system. In the presence of thiamine pyrophosphate, pyruvate, coenzyme A, and Mg2+, E3 inactivation by BrCH2CONHPhAsO was selective (coenzyme A binding was unaffected) and stoichiometrically related to PD complex inactivation, indicating that a complement of E3 is necessary for full complex activity. The activity of the pyruvate dehydrogenase (E1) component was unaltered by BrCH2-CONHPhAsO in both systems. On inhibition of the PD complex with BrCH2CONHPhAsO, the reagent mediated interchain cross-linking between E2 and about half of the E3 subunits. A marked change occurred in the quaternary structure of the PD complex, with some E1 and E3 subunits being dissociated from the E2 core. The mechanism outlined by Stevenson et al. [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189] for the inhibition of the PD complex by BrCH2CONHPhAsO must be revised on the basis of these findings. E3 is only partially modified by delivery of the bromoacetyl moiety of the bifunctional reagent (covalently attached to lipoyl residues of E2 through dithioarsinite bonds) into the active site of bound E3. The inhibition of E3, dissociated from the PD complex during cross-linking, likely occurs via direct interaction of the free enzyme with BrCH2CONHPhAsO by initial dithioarsinite modification of the reduced active-site disulfide followed by alkylation of a nearby residue.

Arsenicals↗
PubMed1978

Serum and platelet lipoamide dehydrogenase in Friedreich's ataxia.

Pyruvate dehydrogenase (PDH), alpha-keto glutarate dehydrogenase (alpha-KGDH) and lipoamide dehydrogenase (LAD) were measured in platelets of 11 patients with typical Friedreich's ataxia and 10 normal control subjects. Serum LAD was also evaluated in the same patients. No statistically significant changes were found in platelets for the group as a whole, although some patients had low values (more than one standard deviation below control mean). Serum LAD was significantly reduced in the patients with Friedreich's ataxia. This was not due to associated diabetes.

Blood Platelets↗