Abstract
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
A
̊
),
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
A
̊
),
and from His-95 to the enolic-O
− of PGH
(2.62±0.02
A
̊
).
In the methylglyoxal synthase–PGH complex, a SSHB
(2.51±0.02
A
̊
)
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.
