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PubMed1991

Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase.

The structure of lipoamide dehydrogenase from Azotobacter vinelandii has been refined by the molecular dynamics technique to an R-factor of 19.8% at 2.2 A resolution. In the final model, the root-mean-square deviation from ideality is 0.02 A for bond lengths and 3.2 degrees for bond angles. The asymmetric unit comprises two subunits, each consisting of 466 amino acid residues and the prosthetic group FAD, plus 512 solvent molecules. The last ten amino acid residues of both chains are not visible in the electron density distribution and they are probably disordered. The operation required to superimpose the two chains forming the dimer is a rotation of exactly 180 degrees with no translation component. The final model shows the two independently refined subunits to be very similar, except for six loops located at the surface of the molecule. The structure of each subunit of the enzyme consists of four domains with the catalytic centre located at the subunit interface. The reactive disulphide bridge, 48-53, is oxidized with S gamma of Cys53 located 3.5 A away from carbon C-4a of the isoalloxazine ring. The side-chain of His450' points its N epsilon 2 towards S gamma of Cys48 and is hydrogen bonded to the carboxylate of Glu455'. The FAD is bound in an extended conformation and the isoalloxazine ring is not completely planar with an angle between the pteridine and the benzene ring of 7.3 degrees in the first subunit and of 12.1 degrees in the second one. The overall folding of lipoamide dehydrogenase is very similar to that of glutathione reductase. However, a comparison of the two enzymes, which have only 26% sequence identity, reveals significant conformational differences. These concern the tertiary as well as the quaternary structure of the two molecules. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the interface domain appear to be differently oriented with respect to the FAD-binding domain by 7.1 degrees and 7.8 degrees, respectively. The interface domain contains, in addition, major changes in tertiary structure. Furthermore, the two subunits forming the dimer appear to be shifted with respect to each other by more than 4 A, when the lipoamide dehydrogenase dimer is compared with that of glutathione reductase. In spite of all these changes at the tertiary and quaternary level the active sites of the enzymes, which occur at the dimer interface, appear to be remarkably similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter↗
PubMed2002

The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis.

Lipoamide dehydrogenase catalyses the NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-(2)H]-NADH was studied anaerobically at 4 degrees C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (k(for) = 1260 s(-)(1), k(rev) = 590 s(-)(1)) and represents the formation of FADH(2).NAD(+), an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [(D)(k(for) + k(rev)) approximately 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (k(obs) = 200 s(-)(1)). This phase represents the intramolecular transfer of electrons from FADH(2) to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH(2). The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH(4). The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (k(for) = 57 s(-)(1), k(rev) = 128 s(-)(1)) isomerization of EH(2) or slow release of NAD(+) before rapid NADH binding and reaction to form EH(4). The mechanism of oxidation of EH(2) by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH(2) shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH(2).NAD(+) complex. This was followed by two phases. The first phase (k(obs) = 750 s(-)(1)), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH(2).NAD(+). The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of approximately 115 s(-)(1) probably represents the overall rate of formation of E(ox) and NADH from EH(2) and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.

Bacterial Proteins↗
PubMed1985

Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes.

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.

Animals↗
PubMed1992

Lipoamide dehydrogenase from Azotobacter vinelandii. The role of the C-terminus in catalysis and dimer stabilization.

The 10 C-terminal residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures of the lipoamide dehydrogenases from Pseudomonas putida and Pseudomonas fluorescens. In these structures, the C-terminus folds back towards the active site and is involved in interactions with the other subunit. The function of the C-terminus of lipoamide dehydrogenase from A. vinelandii was studied by deletion of 5, 9 and 14 residues, respectively. Deletion of the last 5 residues does not influence the catalytic properties and conformational stability (thermoinactivation and unfolding by guanidinium hydrochloride). Removal of 9 residues results in an enzyme (enzyme delta 9) showing decreased conformational stability and high sensitivity toward inhibition by NADH. These features are even more pronounced after deletion of 14 residues (enzyme delta 14). In addition Tyr16, conserved in all lipoamide dehydrogenases sequenced thus far, and shown from the other structures to be likely to be involved in subunit interaction, was replaced by Phe and Ser. Mutation of Tyr16 also results in a strongly increased sensitivity toward inhibition by NADH. The conformational stability of both Tyr16-mutated enzymes is comparable to enzyme delta 9. The results strongly indicate that a hydrogen bridge between tyrosine of one subunit (Tyr16 in the A. vinelandii sequence) and histidine of the other subunit (His470 in the A. vinelandii sequence), exists in the A. vinelandii enzyme. In the delta 9 and delta 14 enzymes this interaction is abolished. It is concluded that this interaction mediates the redox properties of the FAD via the conformation of the C-terminus containing residues 450-470.

Azotobacter vinelandii↗
PubMed1992

pH dependent kinetic studies of lipoamide dehydrogenase catalysis.

1. Kinetic studies of lipoamide dehydrogenase and its modified enzymes catalyzing lipoamide oxidoreduction and ancillary reactions at various pH are compared. 2. The asymptotic kinetics of lipoamide oxidoreductions switch between the ping pong and ordered mechanisms by varying pH of the reactions. 3. pH-rate profiles of these reactions are bell-shaped suggesting the participation of 2 ionizable residues with pK values of 6.6 +/- 0.5 and above 8 respectively. 4. The unusually high pK value for the catalytic site histidine is attributed to its involvement in an ion-pair formation. 5. In the absence of the catalytic site histidine, the pH-rate profile for the lipoamide reduction of the photooxidized enzyme is no longer bell-shaped but it is similar to those of the transhydrogenation and NADH-oxidation of the native enzyme. 6. This implies the participation of a low-pK protonated group in these reactions.

Animals↗
PubMed1988

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.

Amino Acid Sequence↗
PubMed1991

The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii.

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, tm = 80 degrees C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass greater than 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.

Amino Acid Sequence↗
PubMed1996

Amplified determination of lipoyl groups by lipoamide dehydrogenase in the presence of oxidized glutathione.

Lipoyl group determination by lipoamide dehydrogenase (NADH: lipoamide oxidoreductase; EC 1.8.1.4) was examined using lipoyl lysine as a substrate. The reaction was monitored by the coupled oxidation of NADH at 340 nm absorbance. Dehydrogenase-mediated NADH oxidation was too slow to be used for the quantification of lipoyl groups in the concentration range 1 to 10 microM. However, when glutathione disulfide (GSSG) was added to the reaction mixture to regenerate the oxidized substrate for the enzyme, NADH oxidation was markedly enhanced. This GSSG-dependent enhancement of NADH oxidation was strongly dependent upon the lipoyl substrate, but was only slightly dependent on the amounts of GSSG without the substrate. In the presence of excess GSSG, NADH oxidation was linearly correlated to the concentration of lipoyl lysine up to 10 microM; this assay is suitable for determining micromolar concentrations of the lipoyl moiety.

Dihydrolipoamide Dehydrogenase↗
PubMed1992

The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.

Amino Acid Sequence↗
PubMed1985

Reactions of lipoamide dehydrogenase and glutathione reductase with arsonic acids and arsonous acids.

Lipoamide dehydrogenase reacts irreversibly with arsonous acids, RAs(OH)2, and arsonic acids, RAs(O)(OH)2, to form enzyme-inhibitor complexes. The formation of inactive enzyme requires NADH and is kinetically first order in the presence of excess arsonous acid. The second-order rate constant for formation of the enzyme-inhibitor complex was 545 min-1 M-1 for phenylarsonous acid, C6H5As(OH)2, and 5640 min-1 M-1 for methanearsonous acid, CH3As(OH)2. The kinetics of formation of inactive enzyme in the presence of arsonic acids was found to obey a rate law predicted by a two-step mechanism in which a rate-limiting reduction of an arsonic acid to the corresponding arsonous acid by reduced enzyme, E(SH)2, preceded formation of an inactive binary complex of reduced enzyme and arsonous acid: ES2 + NADH + H+ = E(SH)2 + NAD+; E(SH)2 + RAs(O)(OH)2 = ES2 + RAs(OH)2 + H2O; and E(SH)2 + RAs(OH)2 = ES2AsR + 2H2O. GSSG reductase reacts reversibly with C6H5As(OH)2 to form an inactive binary addition compound in the presence of NADPH. The value of the association constant for formation of enzyme inhibitor complex at pH 7.0 was 119 M-1. The initial rate of the GSSG reductase-catalyzed oxidation of NADPH by GSSG was insensitive to MeAs(OH)2. The kinetics of inhibition of GSSG reductase by arsenite and C6H5As(O)(OH)2 were found to obey the rate law described for lipoamide dehydrogenase and arsonic acids. GSSG reductase catalyzed the oxidation of NADPH by p-arsanilic acid. The initial rate of oxidation of NADPH was linearly dependent on enzyme concentration. The turnover number for GSSG reductase with p-arsanilic acid as an oxidant was 0.13 mol NADPH mol FAD-1 min-1.

Arsanilic Acid↗
PubMed1974

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart.

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.

Amino Acid Sequence↗
PubMed1988

The nucleotide sequence of the LPD1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae: comparison between eukaryotic and prokaryotic sequences for related enzymes and identification of potential upstream control sites.

The complete nucleotide sequence of the LPD1 gene, which encodes the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Saccharomyces cerevisiae, has been established. The flanking region 5' to the LPD1 gene contains DNA sequences which show homology to known control sites found upstream of other yeast genes. The primary structure of the protein, determined from the DNA sequence, shows strong homology to a group of flavoproteins including Escherichia coli lipoamide dehydrogenase and pig heart lipoamide dehydrogenase. The amino acid sequence also reveals the presence of a potential targeting sequence at its N-terminus which may facilitate transport to and entry into mitochondria.

Amino Acid Sequence↗
PubMed1993

Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties.

The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.

Amino Acid Sequence↗
PubMed2001

Purification and characterization of an enzyme from Mycobacterium sp. Pyr-1, with nitroreductase activity and an N-terminal sequence similar to lipoamide dehydrogenase.

Mycobacterium sp. Pyr-1 produces an enzyme with nitroreductase activity that reduces 1-nitropyrene and 4-nitrobenzoic acid to the corresponding aromatic amines. This enzyme was constitutive and required NADH; and its activity was enhanced by FAD. It was inhibited by antimycin A, dicumarol, and o-iodosobenzoic acid; and it was inactivated by ammonium sulfate precipitation. After purification to homogeneity, the protein produced a single band on native and SDS-polyacrylamide gels and had a single amino-terminal sequence. The N-terminal amino acid sequence was identical to the corresponding sequences of the lipoamide dehydrogenases of M. leprae, M. tuberculosis and Corynebacterium glutamicum. The amino-terminal sequence was also similar to lipoamide dehydrogenases from M. smegmatis and several other bacteria. The amino acid sequence of an internal peptide (12 of 13 amino acids) was nearly identical to the corresponding sequences of lipoamide dehydrogenases from M. leprae and M. tuberculosis and was similar to those of C. glutamicum, Streptomyces coelicolor and S. seoulensis. The data show that a unique lipoamide dehydrogenase in Mycobacterium sp. Pyr-1, which differs from classic (Type I) bacterial nitroreductases, reduces aromatic nitro compounds to aromatic amines.

Amino Acid Sequence↗
PubMed1976

Lipoamide dehydrogenase in serum: a preliminary report.

Lipoamide dehydrogenase was identified in serum and the optimal conditions for its assay at 30 degrees C were defined. The pH optimum in tris(hydroxymethyl)aminomethane buffer is 7.8, and activity is inhibited if buffer concentration exceeds 100 mmol/liter. Saturating concentrations of the substrates NAD+ and lipoamide are 3 mmol/liter and 5 mmol/liter, respectively. Activity is decreased eightfold when lipoic acid is substituted for lipoamide. Activity is linearly related to enzyme concentration up to limiting absorbance change of 0.300 at 340 nm, and both within-day and day-to-day precision are satisfactory. Data suggest a normal range (2 SD) of 3-19 kU/liter. The highest value measured in serum was 473 kU/liter. A correlation with direct bilirubin concentrations (r equals 0.435, P less than 0.01) was found.

Bilirubin↗
PubMed1991

Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1978

Lipoamide dehydrogenase in Friedreich's ataxia fibroblasts.

Lipoamide dehydrogenase was measured in cultivated skin fibroblasts from twelve patients with Friedreich's ataxia and nine normal controls. No difference in specific activity, subcellular distribution and Vmax or Km was observed between patients and controls.

Cells, Cultured↗