Multinuclear NMR study of enzyme hydration in an organic solvent

Christopher S. Lee, Michael T. Ru, Mathias Haake, Jonathan S. Dordick, Jeffrey A. Reimer, Douglas S. Clark

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer's physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by 17O- and 2H-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state 2H-NMR spectra of enzyme- bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.

Original languageEnglish
Pages (from-to)686-693
Number of pages8
JournalBiotechnology and Bioengineering
Volume57
Issue number6
DOIs
StatePublished - 20 Mar 1998
Externally publishedYes

Keywords

  • Enzyme hydration
  • NMR spectroscopy
  • Organic solvents
  • Subtilisin

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