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Critical hydrogen bonds and protonation states of pyridoxal 5′-phosphate revealed by NMR

Hans-Heinrich Limbach, Monique Chan-Huot, Shasad Sharif, Peter M. Tolstoy, Ilya G. Shenderovich, Gleb S. Denisov, Michael D. Toney
Biochimica et biophysica acta 2011 v.1814 no.11 pp. 1426-1437
aqueous solutions, aspartate transaminase, aspartic acid, correlation, enzymatic reactions, enzymology, histidine, hydrogen bonding, models, nitrogen, nuclear magnetic resonance spectroscopy, proteins, pyridoxal, pyridoxal phosphate, schiff bases, solvents, stable isotopes, zwitterions
In this contribution we review recent NMR studies of protonation and hydrogen bond states of pyridoxal 5′-phosphate (PLP) and PLP model Schiff bases in different environments, starting from aqueous solution, the organic solid state to polar organic solution and finally to enzyme environments. We have established hydrogen bond correlations that allow one to estimate hydrogen bond geometries from ¹⁵N chemical shifts. It is shown that protonation of the pyridine ring of PLP in aspartate aminotransferase (AspAT) is achieved by (i) an intermolecular OHN hydrogen bond with an aspartate residue, assisted by the imidazole group of a histidine side chain and (ii) a local polarity as found for related model systems in a polar organic solvent exhibiting a dielectric constant of about 30. Model studies indicate that protonation of the pyridine ring of PLP leads to a dominance of the ketoenamine form, where the intramolecular OHN hydrogen bond of PLP exhibits a zwitterionic state. Thus, the PLP moiety in AspAT carries a net positive charge considered as a pre-requisite to initiate the enzyme reaction. However, it is shown that the ketoenamine form dominates in the absence of ring protonation when PLP is solvated by polar groups such as water. Finally, the differences between acid–base interactions in aqueous solution and in the interior of proteins are discussed. This article is part of a special issue entitled: Pyridoxal Phosphate Enzymology.