Catalytic properties of lipoamide dehydrogenase from Mycobacterium smegmatis.
Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to homogeneity over 60-fold. Of 20 amino acid residues identified at the amino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens lipoamide dehydrogenases, respectively. The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment. Reduction of the enzyme with dithionite results in the appearance of an absorbance shoulder at 530-550 nm, suggesting that reducing equivalents of the two-electron reduced enzyme reside predominantly on the redox-active disulfidedithiol. The kinetic mechanism of the forward (NAD+ reducing) and reverse (NADH oxidizing) reactions proved difficult to study due to severe substrate inhibition by NAD+ and NADH. The rate of lipoamide reduction was found to depend upon the NAD+/NADH ratio, with the reaction being activated at low ratios and inhibited at high ratios. The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kinetics to be performed and revealed that the kinetic mechanism is ping pong. In addition to catalyzing the reversible oxidation of dihydrolipoamide, the enzyme displayed high oxidase activity (30% of the lipoamide reduction rate), hydrogen and t-butyl peroxide reductase activity (10% of the lipoamide reduction rate), and both naphthoquinone and benzoquinone reduction (approximately 200% of the lipoamide reduction rate). The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone. The lipoamide-reducing reaction was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions.
Marcinkeviciene
J
J
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Blanchard
J S
JS
D000595:Amino Acid Sequence; D018053:Arsenites / Q000494:pharmacology; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D018053:Arsenites; D013439:Sulfhydryl Reagents; D009243:NAD; D008058:Dihydrolipoamide Dehydrogenase; C015001:arsenite