CRYSTALLOGRAPHIC STUDIES ON THE STEREOSPECIFICITY AND ACCESSIBILITY OF THE a-CHYMOTRYPSIN ACTIVE SITE
Adelia Razeto1, Victor S. Lamzin1, Boris Galunsky2, Volker Kasche2 and Keith Wilson3
1
European Molecular Biology Laboratory (EMBL) Hamburg Outstation,
c/o DESY, Notkestrasse
85, 22603 Hamburg, Germany
2Department of
Biotechnology II, Technical University of Hamburg-Harburg, 21071
Hamburg, Germany
3 Department of
Chemistry, University of York, Heslington, York, Y01 5DD, U.K.
Enzymes are, in general, stereoselective: their active sites are able to distinguish between mirror-image substrates but bind and/or process one enantiomer much better than the other. a-Chymotrypsin and other enzymes of the serine proteinase family are selective towards L-polypeptides. Their stereoselectivity can be modulated by changing the temperature or the solvent hydrophobicity [1]. In particular, kinetic studies indicate that for some substrates there can be an inversion of stereospecificity at selected temperatures. To address the question of how the active site can bind substrates with opposite chirality and why it has more favourable interactions with the L-enantiomer, a comparative crystallographic study of -chymotrypsin complexed with Nboc-L/D-TyrOMe and other substrates is being carried out.
Firstly X-ray data were collected at cryogenic temperature (100K) for the crystals of native -Chymotrypsin. The space group is P21 (a = 43.8, b = 77.5, c = 62.8 A and b= 106.3o) and there are two molecules in the asymmetric unit. The diffraction extends up to 1.3 A resolution, a considerable improvement compared to 1.67 A, the highest resolution reported in the past [2]. The structure has been solved using molecular replacement.
Obtaining crystal structures of complexes with NBoc-L/D-TyrOMe has not proved straightforward. Possible reasons could be the tight crystal packing with restricted accessibility of the active site or special properties of this particular substrate with its bulky and apolar terbutyl group being too large to diffuse through the crystal.
The crystals have first been tested for the accessibility of the active site to PMSF, containing a bulky aromatic group. This inhibitor forms a specific covalent bond to the oxygen of the catalytic serine. Diffraction data were collected to 2.2 A resolution. Already after a few cycles of refinement he sA weighted density maps showed clear density for the inhibitor. Thus the a-chymotrypsin active site in this crystal form is accessible. Then it has been investigated whether non-covalent complexes with aromatic substrates can be obtained. Crystals were soaked in a solution of N-formyl-L-Phe (whose complex with a-chymotrypsin had previously been reported [3]) and diffraction data collected to 2.0 A resolution. A substrate with a slightly longer, acetyl N-terminal protection group, was then tried and data collected to 1.64 A resolution. For both complexes, electron density in the active site clearly indicated the binding and location of the substrates though they were probably only partially occupied. This confirms that in this crystal form the active site can also bind substrates with lower affinity than PMSF.
The results obtained provide
deeper understanding of the fine details of the mode of binding
of stereospecific substrates to the a-chymotrypsin
active site. Detailed analysis will be presented.