Intermediates in the catalytic action of lipoyl dehydrogenase (diaphorase)

Massey, Vincent; Gibson, Quentin H.; Veeger, C.

doi:10.1042/bj0770341

Cited by 235 publications

(107 citation statements)

References 19 publications

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“…In both directions in the absence of added electron acceptors, O 2 is reduced by LADH to superoxide ("oxidase reaction" of LADH), the latter being a reactive oxygen species (ROS), which is then partly dismutated to H 2 O 2 and O 2 [3][4][5][6]. Analogous mechanism to ROS generation by LADH is the diaphorase activity of the enzyme [7], where LADH reduces via NADH various ions or organic molecules as artificial substrates [8][9][10]. The pH optimum of the diaphorase/ROS-generating activity is below 6 (4.8-5.7) [3,8,10,11], while that of the reverse reaction is 6.5-7.3 [8,10].…”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Ambrus

Ádám-Vizi

2013

Archives of Biochemistry and Biophysics

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Human dihydrolipoamide dehydrogenase (LADH, E3) is a component in the pyruvate-, alpha-ketoglutarate-and branched-chain ketoacid dehydrogenase complexes and in the glycine cleavage system. The pathogenic mutations of LADH cause severe metabolic disturbances, called E3 deficiency that often involve cardiological and neurological symptoms and premature death. Our laboratory has recently shown that some of the known pathogenic mutations augment the reactive oxygen species (ROS) generation capacity of LADH, which may contribute to the clinical presentations. A recent report concluded that elevated oxidative stress generated by the above mutants turns the lipoic acid cofactor on the E2 subunits dysfunctional. In the present contribution we generated by molecular dynamics (MD) simulation the conformation of LADH that is proposed to be compatible with ROS generation. We propose here for the first time the structural changes, which are likely to turn the physiological LADH conformation to its ROS-generating conformation. We also created nine of the pathogenic mutants of the ROS-generating conformation and again used MD simulation to detect structural changes that the mutations induced in this LADH conformation. We propose the structural changes that may lead to the modulation in ROS generation of LADH by the pathogenic mutations.

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Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Ambrus

Ádám-Vizi

2013

Archives of Biochemistry and Biophysics

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“…In vitro, DLDH can act as a diaphorase (Massey, et al, 1960) that is capable of transferring electrons from NADH to electron acceptors such as cytochrome c (Igamberdiev, et al, 2004) and ubiquinone (Olsson, et al, 1999, Xia, et al, 2001, and to electron-accepting dyes such as 2,6-dichlorophenolindophenol (DCPIP) (Patel, et al, 1995) and nitroblue tetrazolium (NBT) (Scouten andMcManus, 1971, Sokatch, et al, 1981). While DLDH itself may be a source of reactive oxygen species (Bando and Aki, 1991, Gazaryan, et al, 2002, Sreider, et al, 1990, Tahara, et al, 2007, it is also capable of scavenging nitric oxide (Igamberdiev, et al, 2004) and can serve as an antioxidant by protecting other proteins against oxidative inactivation by 4-hydroxyl-2-nonenal (Korotchkina, et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

Thangthaeng

Forster

2008

Mechanisms of Ageing and Development

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Brain energy metabolism is increased during postnatal development and diminished in neurodegenerative diseases linked to senescence. The objective of this study was to determine if these conditions could involve postnatal or senescence-related shifts in activity or expression of dihydrolipoamide dehydrogenase (DLDH), a key mitochondrial oxidoreductase. Rats ranging from 10 to 60 days of age were used in studies of postnatal development, whereas rats aged 5 or 30 months were used in the aging studies. The expression of DLDH was determined by Western blot analysis using anti-DLDH antibodies and DLDH diaphorase activity was measured by an in-gel activity staining method using nitroblue tetrazolium (NBT)/NADH. Activity of DLDH dehydrogenase was measured as NAD + oxidation of dihydrolipoamide. When these measures were considered in separate groups of 10-, 20-, 30-, or 60-day-old rats, all three showed an increase between 10 and 20 days of age. However, dehydrogenase activity of DLDH showed a further, progressive increase from 20 days to adulthood, in the absence of any further change in DLDH expression or diaphorase activity. No age-related decline in DLDH activity or expression was evident over the period from 5 to 30 months of age. Moreover, aging did not render DLDH more susceptible to oxidative inactivation by mitochondria-generated reactive oxygen species (ROS). Taken together, results of the present study indicate that (1) brain DLDH expression and activity undergo independent postnatal maturational increases; (2) Senescence does not confer any detectable change in the activity of DLDH or its susceptibility to inactivation by mitochondrial oxidative stress.

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“…It is reasonable to assume that these are pairs of equal binding sites because the enzyme contains two independent FAD molecules [ 1,3].…”

Section: Difference Spectramentioning

confidence: 99%

Conformational Studies on Lipoamide Dehydrogenase from Pig Heart

Muiswinkel-Voetberg¹,

Veeger

1973

European Journal of Biochemistry

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1. NAD+ forms two pairs of spectrally distinguishable complexes upon binding to oxidized lipoamide dehydrogenase. The shape end magnitude of the spectra is dependent on temperature and pH. The flavin-absorption band at 450 nm is not shifted uniformly. Intrinsic dissociation constants only could be calculated a t 430 nm. I n 30 mM phosphate buffer p H 7.2 a t 25 "C Kid = 50-60 pM, while a t 0 "C Ki = 40 pM for the high-affinity pair; for the low-affinity pair these values are 200-25OpM and 110-13OpM respectively. The high-affinity pair is also present at pH 5.6 a t I = 0.1 and I = 0.2, the values a t 25 "C are respectively 50 pM and 35 pM.2. Computer simulation suggests that the activating effect of NAD+ on the NADH : lipoate reductase activity can be explained by assuming that binding of NAD+ shifts the pK-value of a protonated group a t the active centre from 6.2 to 4.9-5.0. This suggestion is supported by the observation that binding of NAD+ a t the high-affinity site is accompanied by liberation of 0.9 f 0.07 equiv. H+/mol flavin. From titration data it was calculated that for NAD+ binding 3.The results indicate that binding of NAD+ at the high-affinity site, even at partial saturation, shifts the monomer-dimer equilibrium towards the dimer. Furthermore the difference in reduction state upon reducing the enzyme with NADH a t 25 "C and 0 "C is not due to differences in affinity for NAD+, but to conformational changes of the protein around the flavin moiety. It has to be considered that the two active centres of the dimer are not completely independent.K i = 65 pM. reduced enzyme by reaction of arsenite with the reduced disulfide bridge in the active centre, the addition of NADH results in the formation of the four-equivalent reduced form [4]. Upon four-equivalent reduction of the enzyme with NADH in the presence of arsenite a broad absorption band with a maximum a t 750nm appears, giving the enzyme a blue-green color, which was identified to belong to a charge-transfer complex between NAD+ and FADH,. A sulfhydryl group was postulated to be involved in the binding of NAD+ [1,5].Spectral evidence for NAD+ binding comes from the observation that the spectra of the two-equivalent reduced forms obtained after reduction either by reduced lipoamide or NADH are not completely identical, the latter spectrum shows enhanced absorption a t 530 nm. Veeger and Massey [5] observed that NAD+ bound to the two-equivalent reduced enzyme converts the red intermediate into another intermediate with e flat absorption band in the 500 to 600-nm region. which was proposed to be a two equivalent reduced enzyme * NAD+ complex. Veege et al. [6] demonstrated that this intermediate rathe

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Intermediates in the catalytic action of lipoyl dehydrogenase (diaphorase)

Cited by 235 publications

References 19 publications

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

Conformational Studies on Lipoamide Dehydrogenase from Pig Heart

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