NMR Studies of Active‐Site Properties of Human Carbonic Anhydrase II by Using ¹⁵N‐Labeled 4‐Methylimidazole as a Local Probe and Histidine Hydrogen‐Bond Correlations

Abstract

By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed.

The Partner Does Matter: The Structure of Heteroaggregates of Acridine Orange in Water

“…The reported data show that the absorption and florescence properties of AOH + depend in a characteristic way on both π–π aggregation and hydrogen bonding. The latter can be quantitatively characterized even for complex molecular systems in solutions [46,47], on surfaces [48,49], and in solids [50,51] using 15 N-NMR. Thus, 15 N labeled AO exhibits a very useful sensor to study the structure of soft matter.…”

NMR Study of Solvation Effect on the Geometry of Proton-Bound Homodimers of Increasing Size

“…1 The simplest case is intramolecular proton transfer in small molecules in the solid state. [2][3][4][5] The presence of competing H-bonds and solvent complicates the analysis. [6][7][8] Sophisticated theoretical tools are required in order to simulate enzymatic reactions and transporters and receptors activity at the atomic level.…”

Characterization of Pharmaceutical Cocrystals and Salts by Dynamic Nuclear Polarization-Enhanced Solid-State NMR Spectroscopy

“…33,35 A variety of 6 15 N solid-state NMR experiments have also been applied to understand nitrogen protonation states in organic solids and materials [36][37][38][39][40][41] and biological systems. 24,42,43 Unfortunately, the sensitivity of solid-state NMR spectroscopy is intrinsically poor and many of the aforementioned experiments require extremely long experiment times when samples have 15 NMR experiments on natural abundance samples typically require many hours or even days of signal averaging obtain spectra with adequate signal-to-noise ratio (SNR). 22 Previous 1 H-15 N scalar APT/J-resolved experiments on a cocrystal required more than 4 days of experiment time.…”