1 research outputs found
Imidazole C‑2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
We present a mass spectrometric method
for analyzing protein structure
and function, based on the imidazole C-2 or histidine C<sup>ε1</sup> hydrogen/deuterium (H/D) exchange reaction, which is intrinsically
second-order with respect to the concentrations of the imidazolium
cation and OD<sup>–</sup> in D<sub>2</sub>O. The second-order
rate constant (<i>k</i><sub>2</sub>) of this reaction was
calculated from the pH dependency of the pseudo-first-order rate constant
(<i>k</i><sub>φ</sub>) obtained from the change in
average mass [Δ<i>M</i><sub>r</sub> (0 ≤ Δ<i>M</i><sub>r</sub> < 1)] of a peptide fragment containing
a defined histidine residue at incubation time (<i>t</i>) such that <i>k</i><sub>φ</sub> = −[lnÂ(1
– Δ<i>M</i><sub>r</sub>)]/<i>t</i>. We preferred using <i>k</i><sub>2</sub> rather than <i>k</i><sub>φ</sub> because <i>k</i><sub>2</sub><sup>max</sup> (maximal value
of <i>k</i><sub>2</sub>) was empirically related to p<i>K</i><sub>a</sub> as illustrated with a Brønsted plot [log <i>k</i><sub>2</sub><sup>max</sup> = −0.7p<i>K</i><sub>a</sub> + α (α
is an arbitrary constant)], so that we could analyze the effect of
structure on the H/D exchange rate in terms of logÂ(<i>k</i><sub>2</sub><sup>max</sup>/<i>k</i><sub>2</sub>) representing the deviation of <i>k</i><sub>2</sub> from <i>k</i><sub>2</sub><sup>max</sup>. In the catalytic site of bovine ribonuclease
A, His12 showed a change in logÂ(<i>k</i><sub>2</sub><sup>max</sup>/<i>k</i><sub>2</sub>) much larger than that of His119 upon binding with cytidine 3′-monophosphate,
as anticipated from the X-ray structures and the possible change in
solvent accessibility. However, we need to consider the hydrogen bonds
of the imidazole group with nondissociable groups to interpret an
extremely slow H/D exchange rate of His48 in a partially solvent-exposed
situation