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Subangstrom Crystallography Reveals that Short Ionic Hydrogen Bonds, and Not a His-Asp Low-Barrier Hydrogen
 

Summary: Subangstrom Crystallography Reveals that Short Ionic
Hydrogen Bonds, and Not a His-Asp Low-Barrier Hydrogen
Bond, Stabilize the Transition State in Serine Protease
Catalysis
Cynthia N. Fuhrmann, Matthew D. Daugherty, and David A. Agard*
Contribution from the Howard Hughes Medical Institute and Department of Biochemistry and
Biophysics, UniVersity of California, San Francisco, 600 16th Street, Box 2240,
San Francisco, California 94143-2240
Received November 13, 2005; E-mail: agard@msg.ucsf.edu
Abstract: To address questions regarding the mechanism of serine protease catalysis, we have solved
two X-ray crystal structures of R-lytic protease (RLP) that mimic aspects of the transition states: RLP at pH
5 (0.82 resolution) and RLP bound to the peptidyl boronic acid inhibitor, MeOSuc-Ala-Ala-Pro-boroVal
(0.90 resolution). Based on these structures, there is no evidence of, or requirement for, histidine-flipping
during the acylation step of the reaction. Rather, our data suggests that upon protonation of His57, Ser195
undergoes a conformational change that destabilizes the His57-Ser195 hydrogen bond, preventing the
back-reaction. In both structures the His57-Asp102 hydrogen bond in the catalytic triad is a normal ionic
hydrogen bond, and not a low-barrier hydrogen bond (LBHB) as previously hypothesized. We propose
that the enzyme has evolved a network of relatively short hydrogen bonds that collectively stabilize the
transition states. In particular, a short ionic hydrogen bond (SIHB) between His57 N 2 and the substrate's
leaving group may promote forward progression of the TI1-to-acylenzyme reaction. We provide experimental

  

Source: Agard, David - Department of Biochemistry and Biophysics, University of California at San Francisco

 

Collections: Biotechnology; Biology and Medicine