HIGH-RESOLUTION STRUCTURE OF THE PRE-HYDROLYSIS STATE OF P21RAS COMPLEXED WITH GTP:
NEW INSIGHTS INTO THE ROLE OF WATER MOLECULES IN THE INTRINSIC AND GAP ACTIVATED GTP HYDROLYSIS REACTION OF RAS LIKE PROTEINS

Axel J. Scheidig, Christoph Burmester, and Roger S. Goody

Max-Planck Institute for Molecular Physiology Department of Physical Biochemistry Rheinlanddamm 201 44139 Dortmund Germany, e-mail: scheidig@mpi-dortmund.mpg.de

The structure of the complex between p21ras and its natural substrate GTP has been determined by monochromatic X-ray diffraction methods at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing to stabilize the resulting Michaelis complex. The structure of this complex differs from that of p21ras·GppNHp with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the -phosphate group. The structure of the p21ras·GDP complex arising from hydrolysis of GTP in crystals of p21ras·GTP as also been determined at 2.0 A. The results obtained suggest the possibility of a proton shuffling mechanism between the attacking water molecule and an oxygen of the g-phosphate group in the basal GTPase mechanism, but arguments are presented which render this mechanism unlikely for the GAP-activated GTPase. Comparison of the p21ras·GTP ground state structure with that of transition tate analog structures from other work allows a detailed description of events leading to the formation of the transition state in an associative mechanism for the GAP-activated p21ras GTPase. The talk will cover the techniques used for time-resolved crystallography on p21ras and the biological findings: photolysis, data collection (Laue method and monochromatic) at room temperature and cryo- trapping.