Dihydrolipoamide reductase

Three enzyme activities forming a relatively stable complex participate in the overall reaction (Fig. 22) -ketoacid dehydrogenase, dihydrolipoamide acyl-transferase and the flavoprotein dihydrolipoamide reductase. Thiamine pyrophosphate (D 10.4.5), lipoic acid (Table 16) and coenzyme A (D 11) are involved. Acids shortened by one C-atom and activated by binding to CoA are the final products. The activated bond is formed during the dehydrogenation of intermediate I by formation of the acylated lipoic acid (intermediate II). The last steps are transfer of the acyl moiety to coenzyme A and regeneration of the lipoic acid. 

DIHYDROLIPOAMIDE DEHYDROGENASE. GLUTATHIONE REDUCTASE (EC 1.6.4.2) (GR) PUTATIVE FLAVOPROTEIN C26F1.14C. 

A very interesting application of affinity chromatography to the purification of halophilic enzymes was reported by Sundquist and Fahey (1988). These authors have purified the enzymes bis-y-glu-tamylcysteine reductase and dihydrolipoamide dehydrogenase from H. halohium using immobilized metal ion affinity chromatography in high-salt buffers. 

Relative to the dithiol DTT but also to other monothiols such as 2-mercaptoeth-anol, GSH is a poor stimulator of microsomal deiodinase activity even when tested in the presence of NADPH and glutathione reductase [52,60,61]. Deiodinase activity of isolated microsomes is supported to a limited extent by GSH if tested with low (nM) but not high (/zM) rT3 concentrations or with T4 as the substrate. This low potency of GSH has led investigators to explore other physiological cofactors. As mentioned above, the paucity of cytoplasmic dihydrolipoamide makes it an unlikely candidate despite its unsurpassed potency [52], This is supported by the finding that addition of NADH, the cofactor for lipoamide hydrogenase, does not stimulate deiodinase activity of kidney homogenates unless supplemented with lipoamide [52]. 

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). 

Figure 3-7. Sequence alignment of various enzymes in the flavopro-tein disulfide oxidoreductase family. The sequences of the NADP4-dependent enzymes are the glutathione reductase from E. coli (E-GR), human (H-GR), Pseudomonas aeruginosa (P-GR), mercuric reductase from Staphylococcus aureus (S-MR), P. aeruginosa Tn 501 (P-GR), and trypanothione reductase from Trypanosoma congolense (T-TR). The NAD+-dependent enzymes are dihydrolipoamide dehydrogenase from E. coli (E-DD), B. stearothermophilus (B-DD), yeast (Y-DD), and human (H-DD). Residue positions marked with an asterisk correspond to those that were targets of site-directed mutagenesis in the text.

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