CATALYTIC PROPERTIES OF LIPOAMIDE DEHYDROGENASE FROM MYCOBACTERIUM-SMEGMATIS

Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to h omogeneity over 60-fold. Of 20 amino acid residues identified at the a mino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens lipoamide dehydro genases, respectively. The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment. Reduction of the en zyme with dithionite results in the appearance of an absorbance should er at 530-550 nm. suggesting that reducing equivalents of the two-elec tron reduced enzyme reside predominantly on the redox-active disulfide -dithiol. The kinetic mechanism of the forward (NAD(+) reducing) and r everse (NADH oxidizing) reactions proved difficult to study due to sev ere substrate inhibition by NAD(+) and NADH. The rate of lipoamide red uction was found to depend upon the NAD(+)/NADH ratio, with the reacti on being activated at low ratios and inhibited at high ratios. The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kin etics to be performed and revealed that the kinetic mechanism is ping pong. In addition to catalyzing the reversible oxidation of dihydrolip oamide, the enzyme displayed high oxidase activity (30% of the lipoami de reduction rate), hydrogen and t-butyl peroxide reductase activity ( 10% of the lipoamide reduction rate), and both naphthoquinone and benz oquinone reduction (similar to 200% of the lipoamide reduction rate). The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone. The lipoamide-reducing reac tion was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions. (C) 1997 Aca demic Press.