Dihydrolipoamide dehydrogenase from halophilic archaebacteria.
Dihydrolipoamide dehydrogenase has been discovered in the halophilic archaebacteria for the first time. The enzyme from both classical and alkaliphilic halobacteria has been investigated. (1) The enzyme specifically catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+. Enzymic activity was optimal at 2 M-NaCl and was remarkably resistant to thermal denaturation. (2) The relative molecular masses (Mr) of the native enzyme from the various species of halobacteria were determined to be within the range 112000-120000. (3) The enzyme exhibited a hyperbolic dependence of catalytic activity on both dihydrolipoamide and NAD+ concentrations. From these steady-state kinetic measurements the dissociation constant (Ks) of dihydrolipoamide was determined to be 57 (+/- 5) microM. (4) The enzyme was only susceptible to inactivation by iodoacetic acid in the presence of its reducing ligands, dihydrolipoamide or NADH. The rate of inactivation followed a hyperbolic dependence on the concentration of dihydrolipoamide, from which the Ks of this substrate was calculated to be 55 (+/- 7) microM. Together with the steady-state kinetic data, the pattern of inactivations is consistent with the involvement in catalysis of a reversibly reducible disulphide bond, as has been found in dihydrolipoamide dehydrogenase from non-archaebacterial species. In eubacterial and eukaryotic organisms, dihydrolipoamide dehydrogenase functions in the 2-oxo acid dehydrogenase complexes. These multienzyme systems have not been detected in the archaebacteria, and, in the context of this apparent absence, the possible function and evolutionary significance of archaebacterial dihydrolipoamide dehydrogenase are discussed.
D001105:Archaea / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology*; D002850:Chromatography, Gel; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006217:Halobacterium / Q000201:enzymology*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008970:Molecular Weight; D013696:Temperature
D007461:Iodoacetates; D008058:Dihydrolipoamide Dehydrogenase; D019807:Iodoacetic Acid