title: Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations.
abstract: Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P(i) by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10(5) compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10(5) compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.
authors.affiliation: Department of Biophysics, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Rudack
authors.forename: Fei
authors.initials: F
authors.lastname: Xia
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schlitter
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
mesh_terms: D002384:Catalysis; D003198:Computer Simulation; D020690:GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D010100:Oxygen / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D013055:Spectrophotometry, Infrared / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013816:Thermodynamics; D014965:X-Rays; D018631:ras Proteins / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; D008024:Ligands; D010710:Phosphates; D011522:Protons; D006160:Guanosine Triphosphate; D018631:ras Proteins; D008274:Magnesium; D010100:Oxygen