TRANSITION-STATE STRUCTURE OF THE SOLVOLYTIC HYDROLYSIS OF NAD(+)

The transition state structure has been determined for the pH-independ ent solvolytic hydrolysis of NAD(+). The structure is based on kinetic isotope effects (KIEs) measured for NAD(+)'s labeled in various posit ions of the ribose ring and in the leaving group nitrogen. The KIEs fo r reactions performed at 100 degrees C in 50 mM NaOAc (pH 4.0) were as follows: 1-N-15, 1.020 +/- 0.007; 1'-C-14, 1.016 +/- 0.002; [1-N-15,1 '-C-14], 1.034 +/- 0.002; 1'-H-3, 1.194 +/- 0.005; 2'-H-3, 1.114 +/- 0 .004; 4'-H-3, 0.997 +/- 0.001; 5'-H-3, 1.000 +/- 0.003; 4'-O-18, 0.988 +/- 0.007. The transition state structure was determined using bond e nergy/bond order vibrational analysis to predict KIEs for trial transi tion; state models. The structure that most closely matches the experi mental KIEs defines the transition state. A structure interpolation me thod was developed to generate trial transition state structures and t hereby systematically search reaction coordinate space. Structures are generated by interpolation between reference structures, reactant NAD (+) and a hypothetical {ribo-oxocarbenium ion plus nicotinamide} struc ture. The point in reaction coordinate space where all the predicted K IEs matched the measured ones was considered to locate the transition state structure. This occurred when the residual bond order to the lea ving group nicotinamide, n(LG,TS), was 0.02 (bond length = 2.65 Angstr om) and the bond order to the approaching nucleophile, n(Nu,TS), was 0 .005 (3.00 Angstrom). Thus, bond-breaking and bond-making in this A(N) D(N) reaction are asynchronous, and the transition state has a highly oxocarbenium ion-like character.