A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein

摘要

The hydrolysis of nucleoside triphosphates by enzymes is used as a regulation mechanism in key biological processes. Here, the GTP hydrolysis of the protein complex of Ras with its GTPase-activating protein is monitored at atomic resolution in a noncrystalline state by time-resolved FTIR spectroscopy. At 900 ms, after the attack of water at the γ-phosphate, there appears a H_2PO_4~- intermediate that is shown to be hydrogen-bonded in an eclipsed conformation to the β-phosphate of GDP. The H_2PO_4~- intermediate is in a position where it can either reform GTP or be released from the protein in 7 s in the rate-limiting step of the GTPase reaction. We propose that such an intermediate also occurs in other GTPases and ATPases.