Structure of the liver alcohol dehydrogenase-NAD^+-pyrazole complex as determined by ^(15)N NMR spectroscopy

Abstract

The structures of the liver alcohol dehydrogenase (LADH)-NAD^+-pyrazole and LADH-NAD+-4-ethylpyrazole complexes were investigated by ^(15)N nuclear magnetic resonance (NMR) spectroscopy. ^(15)N chemical shifts were obtained for ^(15)N-labeled inhibitors and ^(15)N-labeled coenzyme bound in the ternary enzyme complexes. The structures of the two inhibitor complexes appear to be very similar. ^(15)N NMR studies of model pyrazole-zinc chloride complexes were carried out to determine the effect of zinc complexation on pyrazole chemical shifts. The N1 nicotinamide chemical shift of the coenzyme of the LADH-NAD^+-pyrazole complex demonstrates that NAD^+ is converted to a dihydronicotin amide derivative in the complex. The N1 chemical shift of the pyrazole in the ternary complex is consistent with covalent bond formation between pyrazole N1 and the nicotinamide ring of the coenzyme. The N2 chemical shift of the pyrazole in the ternary complex indicates that the nucleus of this nitrogen is about 40 ppm more shielded than those of the N2 nitrogens of typical pyrazoles. Such shielding is expected as the result of direct complexation of N2 to the active-site zinc. Shift comparisons with zinc-pyrazole complexes indicate a high degree of inner-sphere coordination of the pyrazole N2 to the active-site zinc in the ternary complex.

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