STEREOSPECIFICITY OF HYDROGEN-TRANSFER BY THE NADP-LINKED ALCOHOL-DEHYDROGENASE FROM THE THERMOPHILIC BACTERIUM THERMOANAEROBIUM-BROCKII

Class A and class B NAD(H)/NADP(H) coenzyme-dependent dehydrogenases d istinguish between the diastereotopic hydrogens pro-R and pro-S at pos ition 4 of the cofactor. We investigated the stereochemistry of hydrid e transfer in reactions catalyzed by an unusual thermophilic, zinc-con taining, NADP-linked enzyme Thermoanaerobium brockii alcohol dehydroge nase (TBAD). Using proton NMR spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers th e pro-R hydrogen from the pyridine 4 position of the reduced coenzyme. This stercospecificity is stable over (a) a broad range of temperatur es up to 70-degrees-C, (b) different concentrations of the coenzyme (c atalytic or stoichiometric) and (c) a wide scope of substrates. Althou gh NAD+ is not an effective coenzyme for TBAD, NADP+ and its synthetic analogs, 3-acetylpyridine-ADP+ and thio-NADP+, can be used successful ly. (C) Munksgaard 1993.