Format

Send to

Choose Destination
Biochemistry. 2004 May 11;43(18):5246-55.

Allosteric changes in solvent accessibility observed in thrombin upon active site occupation.

Author information

1
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0378, USA.

Abstract

The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1.

PMID:
15122890
DOI:
10.1021/bi0499718
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center