¹⁵N Nuclear Magnetic Resonance Studies of the Liver Alcohol Dehydrogenase-NAD⁺-Pyrazole Complex

Citation

Becker, Nancy Newton (1983) ¹⁵N Nuclear Magnetic Resonance Studies of the Liver Alcohol Dehydrogenase-NAD⁺-Pyrazole Complex. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/nnx8-q333. https://resolver.caltech.edu/CaltechTHESIS:09262018-161343127

Abstract

The structures of the liver alcohol dehydrogenase (LADH)-NAD⁺-pyrazole and LADH-NAD⁺-4-ethylpyrazole complexes were investigated by ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N chemical shifts were obtained for ¹⁵N-labeled inhibitors and ¹⁵N-labeled coenzyme bound in the enzyme ternary complexes. The structures of the two inhibitor complexes were found to be very similar. ¹⁵N chemical shifts of various pyrazole derivatives were determined. ¹⁵N NMR studies of model pyrazole derivative-zinc chloride complexes were carried out to determine the effect of zinc complexation on the pyrazole N2 chemical shift. The N1 nicotinarnide resonance of the coenzyme of the LADH-NAD⁺-pyrazole complex was 96 ppm upfield from that of NAD⁺ in solution and only 13 ppm downfield from that of NADH in solution, demonstrating formation of a derivative of dihydronicotinamide. The ¹⁵N chemical shift of the pyrazole N1 of the ternary complex when compared to other pyrazole derivatives indicated bond formation between pyrazole N1 and the nicotinarnide ring of the coenzyme. The ¹⁵N shift of the pyrazole N1 of the model pyrazole-NAD⁺ adduct, N-benzyl-1,4-dihydro-4-pyrazolylnicotinarnide, was 5.8 ppm downfield from that of N1 of the enzyme ternary complex. The ¹⁵N chemical shift of the pyrazole N2 of the ternary complex compared to pyrazole derivatives in solution was shielded by more than 40 ppm, demonstrating direct complexation of N2 to the active-site zinc. The results of model zinc complex studies indicated 60-100% inner-sphere coordination of the pyrazole N2 to the active-site zinc in the ternary complex.

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