The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins

Structure. 1999 Nov 15;7(11):1311-24. doi: 10.1016/s0969-2126(00)80021-0.


Background: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides.

Results: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) 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 gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique.

Conclusions: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Formycins / chemistry
  • Formycins / metabolism*
  • Hydrolysis
  • Molecular Structure
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Ribonucleotides / chemistry
  • Ribonucleotides / metabolism*
  • Temperature
  • Water


  • Formycins
  • Ribonucleotides
  • Water
  • formycin triphosphate
  • Proto-Oncogene Proteins p21(ras)