Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activity of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle is controlled by ADP and the reaction products, i. e. succinyl-CoA and NADH. ADP activates the alpha-ketoglutarate dehydrogenase component of the complex, whereas NADH inhibits alpha-ketoglutarate dehydrogenase and lipoyl dehydrogenase. In the presence of NADH the kinetic curve of the complex with respect to alpha-ketoglutarate and NAD and the dependence of upsilon versus [NAD] and upsilon versus [Lip (SH)2] in the lipoyl dehydrogenase reaction are S-shaped. In the absence of inhibitor ADP had no activating effect on lipoyl dehydrogenase; however, in the presence of NADH ADP decreases the cooperativity for NAD. The cooperative kinetics of the constituent enzymes of the complex are indicative of its allosteric properties. Isolation of the alpha-ketoglutarate dehydrogenase complex and its lipoyl dehydrogenase and alpha-ketoglutarate dehydrogenase components in a desensitized state confirms their allosteric nature. It is assumed that NADH effects of isolated alpha-ketoglutarate dehydrogenase is due to a shift in the equilibrium between different oligomeric forms of the enzyme.
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PMID:[Regulation of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]. 22 76

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active-site base, was replaced with Ser, Tyr or Phe. Pro451, from X-ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X-ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His-Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH-/FAD. The wild-type enzyme functions according to a ping-pong mechanism in the physiological reaction in which the formation of NADH is rate-limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady-state kinetics using the NAD+ analog 3-acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863-872] and to the inhibition by NADH. The mutations considerably affect turnover. Enzymes with the mutations Pro451----Ala, His450----Phe and His450----Tyr appear to be almost inactive in both directions. Enzyme His450----Ser is minimally active, V at the pH optimum being 0.5% of wild-type activity in the physiological reaction. Rapid reaction kinetics show that for the His450-mutated enzymes the reductive half reaction using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared using reduced 6,8-thioctic acid amide [Lip(SH)2] is rate-limiting and extremely slow when compared to the wild-type enzyme. For enzyme Pro451----Ala it is concluded that the loss of activity is due to over-reduction by Lip(SH)2 and NADH. The Glu455-mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455----Asp more than Glu455----Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction. The rapid reaction kinetics obtained for enzymes Glu455----Asp and Glu455----Gln deviate from the wild-type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Kinetics of wild-type and mutated enzymes. 163 4

Branched-chain alpha-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain alpha-keto acid decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain alpha-keto acid dehydrogenase from bovine kidney and liver was characterized with an independent radiochemical assay for this component. The assay uses the model reaction: R-14CO-S-CoA + Lip-(SH)2 in equilibrium R-14CO-S-Lip-SH + CoA-SH, which is similar to that catalyzed by the transacetylase component of the pyruvate dehydrogenase complex. In this reaction, exogenous dihydrolipoamide substitutes for the protein (E2)-bound dihydrolipoyl moiety, and [1-14C]acyl-CoA synthesized enzymatically is the acyl-CoA substrate. The thioester structure of the reaction product, S-acyldihydrolipoamide, was identified by mass spectrometry, its characteristic absorption at 232-245 nm and by formation of hydroxamate with hydroxylamine. Rates of the E2-catalyzed transacylation reaction with various [1-14C]acyl-CoAs are in the order of [1-14C]isobutyryl-CoA greater than [1-14C] isovaleryl-CoA greater than [1-14C]acetyl-CoA. The activity with acetyl-CoA is 15% of that with isobutyryl-CoA. The E2 activity is strongly inhibited by arsenite. Modification of the covalently bound lipoyl moiety through reductive acylation in the presence of N-ethylmaleimide is without effect on the transacylation reaction. These data, along with results of initial velocity and product inhibition suggest that the model reaction proceeds via a random Bi Bi mechanism. Limited proteolysis of purified bovine liver branched-chain alpha-keto acid dehydrogenase with trypsin results in complete loss of the overall activity catalyzed by the complex. Nonetheless the activity of the E2 component is not affected. The tryptic digestion cleaves E2 subunits (Mr = 52,600) into a major fragment of Mr = 25,700. By contrast, E1 alpha and E1 beta subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.
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PMID:Catalytic and structural properties of the dihydrolipoyl transacylase component of bovine branched-chain alpha-keto acid dehydrogenase. 674 48

In spite of well-known ability of lipoamide/dihydrolipoamide (LipS2NH2/Lip(SH)2NH2) and oxidized/reduced glutathione (GSSG/GSH) couples to scavenge singlet oxygen (1O2), the possible protective effects of these compounds against photodynamical damage by Alphtalocyanine tetrasulfonate (Al-PcS4) were examined. Using erythrocyte glutathione reductase, pig heart lipoamide dehydrogenase and hamster kidney fibroblast culture as model systems, we have found that protective effects of Lip(SH)2NH2 and LipS2NH2 were close to that of azide, far exceeding the effects of GSH and GSSG, and paralleling the rates of Al-PcS4-sensitized photooxidation of these compounds. We have failed to observe a previously described (Devasagayam, T.P.A., et al. (1991) Biochim Biophys. Acta 1088, 409-412) enhancement of damaging action of 1O2 by GSH. These findings point out to the possibility of LipS2NH2/Lip(SH)2NH2 to neutralize the side-effects of photodynamic therapy, and to a minor but definitely protective role of GSH.
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PMID:The protective effects of dihydrolipoamide and glutathione against photodynamic damage by Al-phtalocyanine tetrasulfonate. 911 32

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.
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PMID:Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes. 1156 Apr 83