Article

NMR localization of protons in critical enzyme hydrogen bonds

Department of Chemistry, University of California, Davis, Davis, California, United States
Journal of the American Chemical Society (Impact Factor: 11.44). 09/2007; 129(31):9558-9. DOI: 10.1021/ja0728223
Source: PubMed

ABSTRACT Using N-15 NMR spectroscopy and hydrogen bond correlations, we have localized a mechanistically critical proton in aspartate aminotransferase in microcrystals and aqueous solution. It is in a H-bond between a carboxylate O of Asp222 and the pyridine nitrogen of pyridoxal-5'-phosphate. At neutral pH in water, aspartate and pyridine are unprotonated, but they share a proton in the enzyme. It is shown that such a binuclear base is typical for acid-base interactions in aprotic polar solvents. Active site H-bonds to Asp222 assist protonation of the pyridine nitrogen in the enzyme, which is considered a prerequisite for catalytic activity. We also show that acid-base behavior in enzymes should be modeled using aprotic polar solvents rather than aqueous solutions.

Download full-text

Full-text

Available from: Peter M Tolstoy, Aug 16, 2015
0 Followers
 · 
143 Views
  • Source
    • "Protonation states in aqueous solution were studied in the case of PLP [11] [12] and of model PLP Schiff bases [13]. The latter were also studied by X-ray crystallography [14] and NMR in the solid state [15], by liquid state NMR in polar solution [16] [17] and, finally, in an enzymatic environment, i.e. aspartate aminotransferase (AspAT) [18]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 (15)N 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.
    Biochimica et Biophysica Acta 06/2011; 1814(11):1426-37. DOI:10.1016/j.bbapap.2011.06.004 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: This paper is based on the method of case-based reasoning to build a decision-making system to discuss the study and development of an urban traffic accident rescue under the ITS condition by the sample of its application.
    Intelligent Transportation Systems, 2003. Proceedings. 2003 IEEE; 11/2003
  • [Show abstract] [Hide abstract]
    ABSTRACT: Three cis-dioxovanadium(V) complexes with similar N-salicylidenehydrazide ligands modeling hydrogen bonding interactions of vanadate relevant for vanadium haloperoxidases are studied by (51)V solid-state NMR spectroscopy. Their parameters describing the quadrupolar and chemical shift anisotropy interactions (quadrupolar coupling constant C(Q), asymmetry of the quadrupolar tensor eta(Q), isotropic chemical shift delta(iso), chemical shift anisotropy delta(sigma), asymmetry of the chemical shift tensor eta(sigma) and the Euler angles alpha, beta and gamma) are determined both experimentally and theoretically using DFT methods. A comparative study of different methods to determine the NMR parameters by numerical simulation of the spectra is presented. Detailed theoretical investigations on the DFT level using various basis sets and structural models show that by useful choice of the methodology, the calculated parameters agree to the experimental ones in a very good manner.
    Solid State Nuclear Magnetic Resonance 03/2008; 34(1-2):52-67. DOI:10.1016/j.ssnmr.2008.02.003 · 2.86 Impact Factor
Show more