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The results of steady-state kinetic measurements on the initial rate of the peroxidatic reaction between β-NAD+ and hydrogen peroxide, catalyzed by horse liver alcohol dehydrogenase, at pH 7 are described. A simple sequential mechanism is deduced from graphical analysis of the data plotted according to Eadie-Augustinsson-Hofstee primary plots and the values of the true kinetic parameters KmNAD, KmH2O2 and V are estimated from the corresponding secondary plots. Ethanol has been found to compete with hydrogen peroxide for the same enzyme active site. During the catalytic process a progressive inactivation of the enzyme occurs caused by H2O2. The rate law of this process is quantitatively described at pH 7 both in the absence and in the presence of NAD+. The coenzyme has been found to protect the enzyme against inactivation by H2O2, which oxidized essential cysteine residues. The results obtained from the study of both catalytic and inactivating processes are finally rationalized on the basis of a general mechanistic scheme. [ABSTRACT FROM AUTHOR]

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the enzyme horse liver alcohol dehydrogenase catalyzes a reaction between NAD+ and H2O2. The final isolated product was then called NADX because of its unknown structure. In this paper the results of spectroscopic investigations on this compound are described. They indicate that only the nicotinamide moiety of the original NAD+ molecule was modified by the action of hydrogen peroxide. From the ¹H and 13C nuclear magnetic resonance spectra of NADX the following structure was deduced: adenosine(5′)diphospho(5)-β-D-ribose-NH-CH=C(CHO)-CONH2. This structure is consistent with both ultraviolet and reactivity measurements performed on NADX. A tentative mechanism for the whole peroxidatic reaction pathway leading to NADX is finally proposed. [ABSTRACT FROM AUTHOR]

The main results obtained during the investigation of the peroxidatic reaction between NAD+ and H2O2, catalyzed by horse liver alcohol dehydrogenase, are here presented. 1. As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the reaction can be followed spectrophotometrically around 300 nm. It consists of a biphasic pattern: the faster step reflects the enzyme-catalyzed formation of a NAD+ derivative, here called ‘compound I’ (ε300 = 15000 M-1 cm-1), from the reagents NAD+ and H2O2. The slower step is enzyme independent and consists of at least two distinct processes, each of them leading independently to the disappearance of the intermediate compound I. These two processes are competitive and conditions can be selected such that either one or the other occurs preferentially: one of them leads to the formation of NADX (ε300 = 23000 M-1 cm-1), the final stable product of the overall reaction, which can be recovered quantitatively only if catalase is added soon after the end of the faster enzymatic step. The second process of the slow step is due to further reaction of compound I with excess H2O2 and the final product Y can be recovered almost quantitatively only if catalase is not added after the fast step. 2. A careful re-examination of the stoichiometry of the reaction unequivocally indicated that one molecule of NAD+ is converted to compound I per molecule of H2O2 reacted at the enzyme active site during the fast step. 3. One proton has been found to be released per mole of NAD+ converted to compound I during the enzymatic fast step. This very significant observation allowed us to deduce the complete stoichiometric balance of the overall reaction. 4. An initial activation of the peroxidatic activity of horse liver alcohol dehydrogenase, followed by a slow progressive decrease of the same activity, was observed when the enzyme was preincubated with 1 mM EDTA. This behaviour has been interpreted as being due to the essential role played by the active-site zinc ion in catalysing the peroxidatic reaction. 5. The peroxidatic reaction has been found to be catalyzed also by yeast alcohol dehydrogenase, thus suggesting a possible physiological meaning to this general ability of alcohol dehydrogenases. [ABSTRACT FROM AUTHOR]