NMR localization of protons in critical enzyme hydrogen bonds

Shasad Sharif, Emily Fogle, Michael D. Toney, Gleb S. Denisov, Ilya G. Shenderovich, Gerd Buntkowsky, Peter M. Tolstoy, Monique Chan Huot, Hans Heinrich Limbach

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Abstract

Using 15N 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.

Original languageEnglish (US)
Pages (from-to)9558-9559
Number of pages2
JournalJournal of the American Chemical Society
Volume129
Issue number31
DOIs
StatePublished - Aug 8 2007

ASJC Scopus subject areas

  • Chemistry(all)

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    Sharif, S., Fogle, E., Toney, M. D., Denisov, G. S., Shenderovich, I. G., Buntkowsky, G., Tolstoy, P. M., Huot, M. C., & Limbach, H. H. (2007). NMR localization of protons in critical enzyme hydrogen bonds. Journal of the American Chemical Society, 129(31), 9558-9559. https://doi.org/10.1021/ja0728223