Time-resolved FTIR studies of the GTPase reaction of H-ras p21 reveal a key role for the beta-phosphate

Abstract

FTIR difference spectroscopy has been established as a new tool to study the GTPase reaction of H-ras p21 (Ras) in a time-resolved mode at atomic resolution without crystallization. The phosphate vibrations were analyzed using site specifically 18O-labeled caged GTP isotopomers. One nonbridging oxygen per nucleotide was replaced for an 18O isotope in the alpha-, beta-, or gamma-position of the phosphate chain. In photolysis experiments with free caged GTP, strong vibrational coupling was observed among all phosphate groups. The investigation of Ras*caged GTP photolysis and the subsequent hydrolysis reaction of Ras*GTP showed that the phosphate vibrations are largely decoupled by interaction with the protein in contrast to free GTP. The characteristic isotope shifts allow band assignments to isolated alpha-, beta-, and gamma-phosphate vibrations of caged GTP, GTP, and the liberated inorganic phosphate. The unusually low frequency of the beta (PO2-) vibration of Ras-bound GTP, as compared to free GTP, indicates a large decrease in the P-O bond order. The bond order decrease reveals that the oxygen atoms of the beta (PO2-) group interact much more strongly with the protein environment than the gamma-oxygen atoms. Thereby, electrons are withdrawn from the beta-phosphorus, and thus also from the beta/gamma-bridging oxygen. This leads to partial bond breakage or at least weakening of the bond between the beta/gamma-bridging oxygen and the gamma-phosphorus atom as a putative early step of the GTP hydrolysis. Based on these results, we propose a key role of the beta-phosphate for GTP hydrolysis. The assignments of phosphate bands provide a crucial marker for further time-resolved FTIR studies of the GTPase reaction of Ras.