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Biochemistry. 1996 Aug 27;35(34):11092-7.

Protonation-state dependence of hydrogen bond strengths and exchange rates in a serine protease catalytic triad: bovine chymotrypsinogen A.

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1
National Magnetic Resonance Facility at Madison, Wisconsin, USA. markley@nmrfam.wisc.edu

Abstract

Hydrogen-1 nuclear magnetic resonance spectroscopy was used to measure D/H fractionation factors and the temperature dependence of the rate of hydrogen exchange at two sites in the catalytic triad of chymotrypsinogen (hydrogen bond between aspartate-102 and histidine-57 and hydrogen bond between histidine-57 and serine-195) as a function of the protonation states of the constituent residues. Connectivities in one-dimensional spectra used to assign NMR data were collected at three pH values: pH 9, at which His-57 is neutral and Asp-102 is negatively charged; pH 3.5, at which His-57 is positively charged and Asp-102 is negatively charged; and pH 1, at which His-57 is positively charged and Asp-102 is neutral. The signal from H epsilon 2 of histidine-57 was assigned by reference to 1H-1H NOE connectivities at pH 3.5 to the previously assigned signals from the H epsilon 1 and H delta 2 of the same residue. The D/H fractionation factor, phi, for the hydrogen bond between Asp-102 and His-57 changed from phi = 2 at pH 9 to phi = 0.4 at pH 3.5. From studies of model systems, it may be concluded that a change of phi of this magnitude corresponds to a large increase in hydrogen bond strength. A signal from the hydrogen bond between Ser-195 and His-57 was detected only at the lower pH values studied. The D/H fractionation factor for this hydrogen bond was phi = 0.7 at pH 3.5, indicative of a moderately strong interaction. Data obtained at pH 1 indicate that the hydrogen bond between Asp-102 and His-57 is weakened but that the hydrogen bond between His-57 and Ser-195 persists. The results are consistent with the hypothesis that changes in hydrogen bonding strength serve to lower barriers along the reaction coordinate in the catalytic mechanism. Large pH-dependent changes were found in the activation enthalpy (delta H ++) for exchange with protons from the solvent at the hydrogen bond between aspartate-102 and histidine-57: delta H ++ was approximately 10-12 kcal.mol-1 higher at pH 3.5 than at pH 1 or 9.

PMID:
8780512
DOI:
10.1021/bi961366k
[Indexed for MEDLINE]
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