HISTIDINOL DEHYDROGENASE - O-18 ISOTOPE SHIFT IN C-13 NMR REVEALS THE ORIGIN OF HISTIDINE OXYGENS

The reaction catalyzed by L-histidinol dehydrogenase (EC 1.1.1.23) is the unusual NAD-linked 4-electron oxidation of L-histidinol carried out by way of an unknown aldehyde level intermediate that is covalently linked to the enzyme. Proposals have included an imine derivative of an active site lysine. The 18O isotope shift in 13C NMR was used to investigate the origin of the two oxygens in product histidine. Histidinol dehydrogenase from Salmonella typhimurium was incubated with highly enriched L-[hydroxymethyl-13C]histidinol with NAD in 50% H18OH; an NAD regenerating system was used to maintain high levels of NAD. Direct examination of the product L-[carboxy-13C]histidine in 13C NMR revealed two peaks separated by 0.02 ppm, indicative of incorporation of a single solvent oxygen. The failure to expel the original histidinol oxygen provides evidence against the participation of an imine in the reaction pathway. © 1990, American Chemical Society. All rights reserved.