Enzyme-bound NAD

Another important property of TDPG-oxidoreductase is its reversible dissociation with enzyme bound-NAD+ (30,31). Treatment of the enzyme with p-chloromercuriphenylsulfonate results in the release of enzyme bound NAD+ and formation of two protein subunits. Incubation of these inactive subunits with cysteine and excess NAD+ leads to reassociation of the subunits and complete return of enzymatic activity. Conditions for the isolation of the subunits of molecular weight of about 40,000 were described by Zarkowsky et al. (32). 

From a mechanistic viewpoint this group of enzymes is best described as oxidoreductases where intramolecular hydride transfer is mediated by enzyme bound NAD+ (Table IV). Different enzymatic end products for these enzymes are a consequence of different ways of stabilization of the 4-ulose intermediates or the 4 carbonium ion. The stabilization of the 4-ulose derivative can occur in several ways as shown by the various examples mentioned. During the molecular rearrangement, initiated by the formation of the 4-ulose, the reaction intermediates are held bound to the enzyme until at the final step enzyme-NADH donates the hydrogen to the last intermediate and enzyme-NAD+ releases the end product from the enzyme. 

Mechanism of action of S-adenosylhomoeysteine hydrolase (enzyme) and its inhibition by deoxyadenosine. (a) The enzyme uses enzyme-bound NAD+ to temporarily oxidize substrate and eventually hydrolyze it to adenosine and homocysteine. [Reproduced with permission from R. H. Abeles, Suicide enzyme inactivators. Chem. Eng. News 61(38), 55 (September 19, 1983). 1983 by the American Chemical Society.] (b) Deoxyadenosine, a suicide substrate, is also oxidized by the enzyme with the formation of a ketosugar, which undergoes decomposition, with the product dissociating from the enzyme and leaving the enzyme in the reduced state (NADH). 

In Equation 3, Tj is the tetrahedral intermediate that undergoes oxidation by enzyme-bound NAD+, while T2 is a tetrahedral adduct with water (or hydrox-... 

Nelsestuen and Kirkwood obtained additional evidence in support of the oxidation-reduction mechanism by study of the reaction catalyzed by a highly purified epimerase containing NAD . When treated with sodium borohydride in the presence of UDP-D-glucose, the enzyme-bound NAD was reduced, with a concomitant loss of enzymic activity towards UDP-D-glucose. The reduced enzyme regained practically all of its activity when it was incubated with dTDP-6-deoxy-D-xyfo-hexos-4-ulose ( 4-keto-6-deoxy-D-glucose ), and NADH was simultaneously oxidized with a loss of tritium,... 

Strategies for Transient Kinetic Measurements. To simplify the kinetic behavior of the system, two sets of experimental conditions were selected. In the direction of aldehyde reduction, reaction was carried out under conditions where both aldehyde and NADH were present in large excess relative to the initial enzyme concentration, [E]o. To achieve the limitation of reaction to a single turnover of sites, the reaction was carried out in the presence of the potent inhibitor pyrazole (Pyr). Pyrazole reacts rapidly and quasi-irreversibly to trap enzyme-bound NAD product in the form of a covalent adduct at the 4-position of the nicotinamide ring [Eq. (2)]. 

The existence of such a pKa perturbation finds additional support from kinetic studies (Fig. 4) which unequivocably establish that the displacement of enzyme-bound NAD+ by NADH at pH 8.8 is accompanied by the net uptake of approximately one mole of hydrogen ion from solution per mole of NAD+ displaced (68). 

Palmer and Abeles investigated this interesting problem and found that the enzyme contains one tightly bound NAD per subunit, a result that we have confirmed in our laboratory. The discovery that AdoHcy hydrolase contains tightly bound NAD led Palmer and Abeles to propose the complex mechanisms shown in Fig. 3. The mechanism proposed by Palmer and Abeles involves the oxidation of the 3 -hydroxyl group of AdoHcy by enzyme bound NAD. The result... 

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