Short, strong hydrogen bonds on enzymes: NMR and mechanistic studies

A. S. Mildvan, M. A. Massiah, T. K. Harris, G. T. Marks, D. H.T. Harrison, C. Viragh, P. M. Reddy, I. M. Kovach

Research output: Contribution to journalArticlepeer-review

58 Scopus citations


The lengths of short, strong hydrogen bonds (SSHBs) on enzymes have been determined with high precision (±0.05 Å) from the chemical shifts (δ), and independently from the D/H fractionation factors (φ) of the highly deshielded protons involved. These H-bond lengths agree well with each other and with those found by protein X-ray crystallography, within the larger errors of the latter method (±0.2 to ± 0.8 Å) [Proteins 35 (1999) 275]. A model dihydroxynaphthalene compound shows a SSHB of 2.54 ± 0.04 Å based on δ = 17.7 ppm and φ = 0.56 ± 0.04, in agreement with the high resolution X-ray distance of 2.55 ± 0.06 Å. On ketosteroid isomerase, a SSHB is found (2.50 ± 0.02 Å), based on δ = 18.2 ppm and φ = 0.34, from Tyr-14 to the 3-O- of estradiol, an analog of the enolate intermediate. Its strength is ∼7 kcal/mol. On triosephosphate isomerase, SSHBs are found from Glu-165 to the 1-NOH of phosphoglycolohydroxamic acid (PGH), an analog of the enolic intermediate (2.55 ± 0.05 Å), and from His-95 to the enolic-O- of PGH (2.62 ± 0.02 Å). In the methylglyoxal synthase-PGH complex, a SSHB (2.51 ± 0.02 Å) forms between Asp-71 and the NOH of PGH with a strength of ≥4.7 kcal/mol. When serine proteases bind mechanism-based inhibitors which form tetrahedral Ser-adducts analogous to the tetrahedral intermediates in catalysis, the Asp...His H-bond of the catalytic triad becomes a SSHB [Proc. Natl Acad. Sci. USA 95 (1998) 14664], 2.49-2.63 Å in length. Similarly, on the serine-esterase, butyrylcholinesterase complexed with the mechanism-based inhibitor m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone, a SSHB forms between Glu-327 and His-438 of the catalytic triad, 2.61 ± 0.04 Å in length, based on δ = 18.1 ppm and φ = 0.65 ± 0.10. Very similar results are obtained with (human) acetylcholinesterase. The strength of this SSHB is at least 4.9 kcal/mol.

Original languageEnglish (US)
Pages (from-to)163-175
Number of pages13
JournalJournal of Molecular Structure
Issue number1-3
StatePublished - Sep 26 2002
Externally publishedYes


  • Nucleic acids
  • Phosphoglycolohydroxamic acid
  • Short, strong hydrogen bonds

ASJC Scopus subject areas

  • Structural Biology
  • Organic Chemistry
  • Physical and Theoretical Chemistry
  • Spectroscopy
  • Atomic and Molecular Physics, and Optics


Dive into the research topics of 'Short, strong hydrogen bonds on enzymes: NMR and mechanistic studies'. Together they form a unique fingerprint.

Cite this