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Unusual Four-Bond Secondary H/D Isotope Effect Supports a Short−Strong Hydrogen Bond between Phospholipase A2 and a Transition State Analogue Inhibitor

A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA2 in the presence of a phosphonate transition state analogue. The cross-modulation of Hε2/H48 and Hδ1/H48 resonances was confirmed by li...

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Bibliographic Details
Published in:Biochemistry (Easton) 2005-03, Vol.44 (12), p.4748-4754
Main Authors: Yuan, Chunhua, Tu, Shengjiang, Gelb, Michael H, Tsai, Ming-Daw
Format: Article
Language:English
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Summary:A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA2 in the presence of a phosphonate transition state analogue. The cross-modulation of Hε2/H48 and Hδ1/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H2O/D2O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA2, the observation that Hδ1/H48 induces a 3-fold larger H/D secondary isotope effect (∼0.15 ppm) on Hε2/H48 than vice versa (∼0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short−strong hydrogen bond formed between the donor Nδ1/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA2 and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi047503s