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Breaking the low barrier hydrogen bond in a serine protease

Breaking the low barrier hydrogen bond in a serine protease The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water‐miscible organic co‐solvent such as N, N‐dimethylformamide (DMF). However, in 50% DMF, the kcat for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the kcat for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397 + 1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 å resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 1808 around the Cβ‐Cγ bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co‐solvents. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Science Wiley

Breaking the low barrier hydrogen bond in a serine protease

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References (42)

Publisher
Wiley
Copyright
Copyright © 1999 The Protein Society
ISSN
0961-8368
eISSN
1469-896X
DOI
10.1110/ps.8.2.410
pmid
10048334
Publisher site
See Article on Publisher Site

Abstract

The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water‐miscible organic co‐solvent such as N, N‐dimethylformamide (DMF). However, in 50% DMF, the kcat for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the kcat for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397 + 1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 å resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 1808 around the Cβ‐Cγ bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co‐solvents.

Journal

Protein Science – Wiley

Published: Jan 1, 1999

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