Solid-State 17O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases

We report preparation, trapping, and solid-state 17O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[17O]­benzoyl-chymotrypsin, trans-o-methoxy-[17O]­cinnamoyl-chymotrypsin, and trans-p-methoxy-[17O]­cinnamoyl-chymotrypsin. We show that both the 17O...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:The journal of physical chemistry. B 2016-11, Vol.120 (43), p.11142-11150
Hauptverfasser: Tang, Aaron W, Kong, Xianqi, Terskikh, Victor, Wu, Gang
Format: Artikel
Sprache:eng
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:We report preparation, trapping, and solid-state 17O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[17O]­benzoyl-chymotrypsin, trans-o-methoxy-[17O]­cinnamoyl-chymotrypsin, and trans-p-methoxy-[17O]­cinnamoyl-chymotrypsin. We show that both the 17O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental 17O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C17O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the 17O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility of 17O as a useful nuclear probe in NMR studies of enzymes.
ISSN:1520-6106
1520-5207
DOI:10.1021/acs.jpcb.6b08798