A transition state analog for two pyruvate metabolizing enzymes, lactate dehydrogenase and alanine dehydrogenase

Abstract

The synthesis of 5-(2-oxalylethyl)-NADH, a NADH derivate with pyruvate covalently attached to the 5 position of the dihydronicotinamide ring over an additional methylene group, was described previously. In the presence of lactate dehydrogenase [pig heart and muscle] [EC 1.1.1.27] the dihydropyridine ring of this coenzyme-substrate analog is oxidized, and the carbonyl function of the side chain is reduced to the corresponding L-hydroxy derivate with a Vmax of 1/3000 of the natural reaction. This reaction is intramolecular as shown by competition experiments with pyruvate. 5-(2-Oxalylethyl)-NADH (pyr-NADH) appears to be a true transition state analog, proving its postulated structure. Pyr-NADH is highly specific for this enzyme as demonstrated by the facts that D-lactate dehydrogenase [Lactobacillus leichmannii] [EC 1.1.1.28] does not catalyze the intramolecular redox reaction, although the substrate moiety of pyr-NADH is reduced in the presence of NADH and when tested with malate dehydrogenase [pig heart] [EC 1.1.1.37], alcohol dehydrogenase [horse liver and yeast] [EC 1.1.1.1], glyceraldehyde phosphate dehydrogenase [rabbit muscle] [EC 1.2.1.12], glycerate dehydrogenase [spinach leaves] [EC 1.1.1.29] and glycerol dehydrogenase [Aerobacter-aerogenes] [EC 1.1.1.6], pyr-NADH is not even oxidized in the presence of the corresponding substrates. A great similarlity between the transition states of the reduction of pyruvate catalyzed by lactate dehydrogenase and alanine dehydrogenase [Bacillus subtilis] [EC 1.4.1.1] could be shown. Alanine dehydrogenase catalyzes the intramolecular redox reaction as well. In the presence of ammonium ions pyr-NADH is transformed to 5-(3-carboxy-3-aminopropyl)-NAD+.