Multifunctionality of lipoamide dehydrogenase: activities of chemically trapped monomeric and dimeric enzymes

Lipoamide dehydrogenase from pig heart exists in monomer-dimer equilibrium. The effect of the state of subunit aggregation on the multifunctionality of lipoamide dehydrogenase was investigated by the use of chemically trapped monomeric and dimeric enzymes. Reductive carboxymethylation with 2-mercaptoethanol-iodoacetate yields the stable monomeric enzyme which has been isolated for structural and kinetic studies. The chemically induced monomerization is accompanied by conformational changes resulting in an increased mobility of flavin-adenine dinucleotide. The chemically trapped monomer shows an enhanced diaphorase activity, a reduced electron transferase activity, and a complete loss in dehydrogenase as well as transhydrogenase activities. The enhanced diaphorase activity is associated with increased catalytic efficiencies and the reversal of an inhibitory NADH effect at high concentrations. Treatment of lipoamide dehydrogenase with dimethyl suberimidate gives amidinated samples containing crosslinked dimer. The crosslinked enzyme exhibits a higher dehydrogenase catalytic efficiency than the noncrosslinked enzyme with different kinetic mechanisms without significantly affecting the kinetic parameters of diaphorase reaction. Although the dimeric structure is intimately associated with the dehydrogenase activity, it does not preclude the diaphorase activity. An altered flavin-adenine dinucleotide environment accompanying monomerization is likely responsible for the enhanced diaphorase activity.