Structure of the liver alcohol dehydrogenase-NAD+-pyrazole complex as determined by nitrogen-15 NMR spectroscopy

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 ternary enzyme complexes. The structures of the two inhibitor complexes appear to be very similar. 15N 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 the NAD+ is converted to a dihydronicotinamide 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.

Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation–π interaction

“…In the wild-type BvHSS structure (PDB entry 4tvb), close proximity of a PUT molecule and an HSP molecule to the nicotinamide ring was similarly observed and was suggested to reflect partial, transient hydride transfers between the respective molecules (Krossa et al, 2016 resolution, the presence and identity of such an adduct remains to be determined by additional experiments. In line with our assumption, NAD adducts have previously been described to be formed in various enzymes between the nicotinamide ring C2N or C4N atoms and both inhibitor compounds (Jacques et al, 2003;Rubach & Plapp, 2003;Benach et al, 1999;Becker & Roberts, 1984) and reaction components (Vö geli et al, 2018;Rosenthal et al, 2014Rosenthal et al, , 2015Choe et al, 2003). NAD(P) adduct formation was thereby proposed to represent an intermediate step in the mechanism of enzymatic hydride transfer (Vö geli et al, 2018;Rosenthal et al, 2014).…”

Heteronuclear NMR studies of Cobalamins. 10. Nitrogen-15 NMR studies of [15N]cyanocobalt corrins

“…37 In addition, 15N NMR spectroscopy has been successfully applied to a number of important problems in macromolecular biochemistry including protein conformation39-42 and interactions in enzyme active sites. [43][44][45][46][47][48][49] Extensive 15N NMR studies of 15Nenriched cyano and isocyanide complexes of low-spin ferrihemes and ferriheme proteins have also been reported by Morishima and co-workers50- 54 and by Goff and co-workers,55 including studies of intact erythrocytes. The cyanide nitrogen resonances of such complexes occur at very low field (as much as 1172 ppm downfield from free CN-), presumably due to paramagnetic effects, and have •On leave from the Department of Chemistry, Indian Institute of Technology, Kanpur, India.…”

NMR-Detected Host–Guest Proton Exchange as a Tool to Explore Surface/Volume Ratios and Fluid Filling of Internal and External Spaces of Porous Solids Containing Surface OH Groups

“…MPz constitutes a drug, which is also called "fomepizole", as it is an inhibitor of alcohol dehydrogenase. 15 At 100 K, MPz forms cyclic H-bonded trimers as found by Xray crystallography. 16 As we were interested in porous SiO 2 materials, which are often used as excipients for drug delivery, 17,18 we have developed and applied the HGPE technique to the study of ordered mesoporous silicas of the MCM-41 19 and SBA-15 types.…”

“…Its boiling point is 477 K and, hence, weight losses due to evaporation are small. MPz constitutes a drug, which is also called “fomepizole”, as it is an inhibitor of alcohol dehydrogenase . At 100 K, MPz forms cyclic H-bonded trimers as found by X-ray crystallography …”