Enzymes lipoamide dehydrogenase

The absence in halobacteria of the oxoacid dehydrogenase complexes creates another puzzle. In most known systems, the role of the enzyme lipoamide dehydrogenase is to reoxidize the lipoic acid that is involved in the oxidation of the oxoacids in the oxoacid dehydrogenase complexes. This enzyme was nonetheless found in H. halobium and purified to homogeneity by Danson et al. (1986). What, then, is its function It is likely that lipoamide dehydrogenase assumes a different role in halobacteria. Another reducing system unique to... 

Scheme 11.33. The oxidative regeneration of the enzyme lipoamide dehydrogenase (EC 1.8.1.4). Flavin adenine dinucleotide (FAD) is reduced in the process to FADH2. EC numbers and some graphic materials provided in this scheme have been taken with permission from appropriate hnks in a URL starting with http //www.chem.qmul.ac.uk/iubmb/enzyme/.

In an early report to a process using three oxidoreductases, namely hydrogenase (ECl.12.2.1), lipoamide dehydrogenase (EC 1.6.4.3) and 20(3-hydroxysteroid dehydrogenase (ECl.1.1.53), a reverse micelle system was used to facilitate stereo- and site-specific reduction of apolar ketosteroids, assisted by the in situ NADH-regenerating enzyme system [61]. 

Enzymes in the cross-linked crystal form are essentially impervious to degradation by exogenous proteases and from autolysis, in the case of CLCs of proteases themselves [5], This stability makes the enzyme-catalyzed preparation of peptides and peptide mimics truly practical [6], Examples will be discussed in more detail in Sec. IV. Further, one could conceive of using multiple enzymes in one-pot reaction systems mimicking natural biosynthetic cascades. Indeed, the application of this concept has been reported for a mixture of lipoamide dehydrogenase and lactate dehydrogenase [19],... 

Cadmium in the body is known to affect several enzymes. It is believed that the renal damage that results in proteinuria is the result of cadmium adversely affecting enzymes responsible for reabsorption of proteins in kidney tubules. Cadmium also reduces the activity of delta-aminolevulinic acid synthetase (Figure 10.3), arylsulfatase, alcohol dehydrogenase, and lipoamide dehydrogenase, whereas it enhances the activity of delta-aminolevulinic acid dehydratase, pyruvate dehydrogenase, and pyruvate decarboxylase. 

Figure 12.18. Output of Pfam search results. Pfam search is performed with amino acid sequence derived from lipoamide dehydrogenase (Schizosaccharomyces pombe). The table for the trusted matches from Pfam-A for pyr redox (pyridine nucleotide disulfide oxidoreductase) and pyr redox dim (pyridine nucleotide disulfide oxidoreductase, dimerization) domains and their alignments (partial) to HMMs ( ->) are shown. The trusted matches from Pfam-B, the potential matches (Thi4 for thiamine biosynthetic enzyme domain), and the bead-on-a-string sketches are not shown. Select the linked domain name to view the functional description of the domain. The HMM alignments are followed by an option button (Align to seed or Align to family) that enables the user to view/save the multiple alignment of each matched family.

Lipoamide dehydrogenase (15, 16) and NADH-cytochrome 65 reductase (17) were covered in detail in the second edition of The Enzymes. The material in these chapters has stood the test of time remarkably well and will only need to be summarized here. 

It is not surprising that enzymes catalyzing such similar chemical reactions should bear striking similarity to one another both structurally and mechanistically. Lipoamide dehydrogenase (34-38), glutathione reductase (39), and thioredoxin reductase (SO, 31) contain, in addition to FAD, a reactive disulfide which is functional in catalysis. These fiavoproteins consist of two identical or near identical polypeptide chains, each with a reactive cystine residue, and two molecules of FAD (31-36). 

The three enzymes are quite specific for their respective pyridine nucleotide substrates. Under conditions normally used for assay, lipoamide dehydrogenase is less than % as active with NADPH as with NADH IS) and thioredoxin reductase is less than 1% as active with NADH as with NADPH 36, Sff). Lipoamide dehydrogenase can transfer electrons to a number of NAD analogs 37). Yeast glutathione reductase is quite specific for NADPH 60), but the erythrocyte enzyme is 20% as active with NADH as with NADPH under the conditions of the standard assay 30,40,61). 

In this section on mechanism and in the section to follow on structure, the comparisons will show that the relationship between lipoamide dehydrogenase and glutathione reductase is more marked than is the relationship of either to thioredoxin reductase. Thus, in catalysis, lipoamide dehydrogenase and glutathione reductase cycle between the oxidized state and a spectrally characteristic state in which the enzyme has accepted two electrons and these are shared between the FAD and the active center disulfide. This intermediate does not seem to be operative in thioredoxin reductase, and in this enzyme the FAD and disulfide interact in a different way. The oxidized forms of these enzsrmes can then be represented as... 

The spectra of oxidized glutathione reductase and of the 2-electron-reduced enzyme are shown in Fig. 1. This red intermediate, which has been shown to be functional in catalysis 39), will be referred to as EH2 designating a half-reduced active center it has also been referred to as F (S3, 53), but this can be confused with oxidized flavin in other nomenclatures. Its spectral characteristics are virtually identical with those of the analogous species of lipoamide dehydrogenase 34, 37, 64)- It has... 

Fia. 2. Anaerobic reduction of pig heart lipoamide dehydrogenase in the presence of arsenite (27) 1, oxidized enzyme plus 1 mM arsenite 2, after 1X> equivalent NADH 3, after 23 equivalents NADH 4, after 33 equivalents NADH and 6, after XADase. 

E. coli lipoamide dehydrogenase has a valine residue preceding the first half-cystine, whereas the eukaryote enzymes have a threonine residue. Chemically this is a relatively conservative change since the side chains of these amino acids are virtually identical in volume. 

Lipoamide dehydrogenase has been isolated from many species and these are listed in Table II (4, S6, 90-115). The mammalian enzymes are of mitochondrial origin. Where tested, the enzyme from eukaryotic sources is isozymic, while that from prokaryotes is a single species (105). There are marked differences in the sensitivity of the enzyme as isolated from various sources to inhibition by excess NADH in the NADH -> lipSaNH reaction and in the relief of that inhibition by NAD+ (27, 99, 105, 106, 108, 109, 116, 117). Attempts to make phylogenetic... 

The spectral characteristics of pig heart lipoamide dehydrogenase reduced anaerobically at pH 7.6 and 25° under the following conditions are virtually identical 1 equivalent (1 mole/mole of enzyme FAD) of... 

The stability of EH2 is very species dependent. All of the above results refer to the pig heart enzyme and, where tested, to other mammalian species. It was initially reported that no long wavelength absorption was observed upon reduction of E. coli enzyme with NADH 109), but reduction by 1 equivalent of NADH or dihydrolipoamide leads to the formation of 25% of the maximal 2-electron-reduced species 108) and similar results are obtained with the Azotobacter enzyme 114)- That this species is the catalytically important one in the E. coli enzyme as well as in the mammalian enzyme has also been demonstrated 50). Reduction with dihydrolipoamide in the rapid reaction spectrophotometer at 2° results in the full formation of EH2 followed by the slow k = 13 min, 1 mAf dihydrolipoamide) four-electron reduction. The spectrum of EHa generated in this way is shown in Fig. 7 and is identical with that of the pig heart enzyme. The 2-electron-reduced form, EHj of lipoamide dehydrogenase of spinach 99) may be somewhat unstable however, spectrally it is difficult to distinguish between instability and formation of the EHa-NADH complex (see above) on the basis of available spectral data. Either phenomenon could lead to inhibition by excess NADH. In glutathione reductase it is possible that the complex can be rapidly reoxidized by glutathione 53). 

The kinetics of the half-reactions for pig heart lipoamide dehydrogenase, i.e., the conversion of enzyme to EHj by NADH or dihydrolipo-amide and the reoxidation of EH by NAD or lipoamide derivatives, have been measured by rapid reaction spectrophotometry (24, 137). Reduction of the enzyme by NADH and reoxidation of EH2 by NAD are complete in the dead time of the instrument which is 3 msec. The rate of reduction of the enzyme by dihydrolipoamide is rate determining in the overall reaction and is 33,000 min" at infinite reductant concentration the same rate is determined by conventional kinetics at infinite concentration of both substrates (24) ... 

A third study of the kinetics of lipoamide dehydrogenase has utilized the enzyme isolated from rat liver (95). At 25°, the temperature of the two previous studies, when dihydrolipoamide was varied at fixed levels of NAD, the double reciprocal plots were concave down. At 37° this behavior was not observed. The detailed studies were carried out at the higher temperature. Rates were measured in both directions at pH 8.0, the pH optimum for the reduction of NAD. Under these conditions, initial velocity patterns for the forward and reverse reactions were a series of parallel lines. The Km for NAD was 0.52 mM, for dihydrolipoamide was 0.49 mAf, for NADH was 0.062 mM, and for lipoamide was... 

The kinetics of the yeast lipoamide dehydrogenase in the direction of NAD+ reduction indicate a bi-bi ping-pong mechanism is operative in this species also (117). If the enzyme from yeast indeed proves to have a tighter EHa-NADH complex than does the mammalian enzyme, product inhibitions studies should show impressive dependence on the fixed substrate (9S). 

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