NDLI: Functional and Structural Roles of the Cys14-Cys38 Disulfide of Bovine Pancreatic Trypsin Inhibitor

Content Provider	PubMed Central
Author	Zakharova, Elena Horvath, Martin P. Goldenberg, David P.
Abstract	The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor (BPTI) lies on the surface of the inhibitor and forms part of the protease binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and x-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58 Å resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical to that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range from 3.5 to 8, decreasing at the more alkaline values, as compared to the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of BPTI and similar inhibitors to resist hydrolysis.
Related Links	http://dx.doi.org/10.1016/j.jmb.2008.07.063
Ending Page	1013
Page Count	16
Starting Page	998
File Format	PDF
ISSN	00222836
e-ISSN	10898638
Journal	Journal of molecular biology
Issue Number	4
Volume Number	382
Language	English
Publisher Date	2008-10-01
Access Restriction	Open
Subject Keyword	Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Structural Biology Molecular Biology Biophysics

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