Heart lipoamide dehydrogenase

A. J. W. G. Visser, H. J. Grande, and C. Veeger, Rapid relaxation processes in pig heart lipoamide dehydrogenase revealed by subnanosecond resolved fluorometry, Biophys. Chem. 12, 35-49 (1980). 

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. 

The free carboxyl groups of pig heart lipoamide dehydrogenase have been determined as 56 per FAD (64). The glutamine plus asparagine content is, by difference, 35. It can then be calculated that the net charge on the protein will depend on the effective pK values of the histidine residues and will vary with pH from -(-5 to —6 per FAD. Amide data for thioredoxin reductase 31) allow a similar calculation The charge can vary from - -2 to —8 per FAD. 

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 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) ... 

The pH optimum of the pig heart lipoamide dehydrogenase in the direction of NAD reduction by dihydrolipoamide is 7.9 (4). In the direction of NADH oxidation by lipoamide the pH optimum is 6.5 (4). In this latter direction there is an absolute requirement for NAD at the pH optimum (S7), but this requirement disappears as the pH is raised (116). It is therefore crucial to be aware of the pH of the measiu ements in comparing kinetic data. 

The total half-cystine content of pig heart lipoamide dehydrogenase is 10 per FAD 6S). The basis for the protein quantitation has been discussed in Section II,C. Older data suggested that there were 2 cystine residues and 6 cysteine residues 83), but more recent data 61, 63) give strong evidence that the active center cystine residue is the only cystine residue. Only 7 thiols react with DTNB under denaturing conditions (dS) however, recalculation of data of Brown and Perham 86), taking... 

Treatment of native pig heart lipoamide dehydrogenase with cupric ion leads to loss of lipoate-linked activities and to a marked increase (10- to 30-fold) in the NADH-DCI activity. Concomitantly there is a drop of 2 in the number of titratable thiols. The action of cupric ion is catalytic 164, 155). Amperometric titration in the presence of urea before and after addition of sulfite indicates that the cupric ion-treated enzyme contains one disulfide in addition to the active center disulfide 155). Sulfite reacts with disulfides as follows ... 

Williams CH, Arscott LD, Shulz GE. 1982. Amino acid sequence homology between pig heart lipoamide dehydrogenase and human erythrocyte glutathione reductase. Proc Natl Acad Sci USA 79 2199-2201. 

Fio. 6. Complex formation between the 2-electron-reduced form of pig heart lipoamide dehydrogenase and NAD 118). 

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