The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

Abstract

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved ''arginine finger'' of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras⅐RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling ''off'' to the signaling ''on'' state with a rate of 3 s ؊1 . The next step is the movement of the ''arginine finger'' into the active site of Ras with a rate of k2 ‫؍‬ 0.8 s ؊1 . Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the ''off'' state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 ‫؍‬ 0.1 s ؊1 , the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the ''arginine finger'' movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate.enzyme catalysis ͉ FTIR spectroscopy ͉ GTPases ͉ phosphate ͉ proteins

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

“…The main changes in coordination result from the intruding arginine finger (Arg789), which displaces the Tyr32 and forms hydrogen bonds with O 13 of the γ-phosphate and to O 11 of the α-phosphate. It has been shown that, once the arginine finger moves into the binding niche, bond cleavage occurs immediately (33). Fig.…”

“…Time-resolved FTIR experiments showed that this bond breakage occurs as soon as the arginine finger intrudes into the binding niche (33). The bond breakage may have at most the same or a lower barrier.…”

“…This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7-22) and experimentally by X-ray crystallography (23-27), electron spin resonance (28-30), and FTIR spectroscopy (5,6,(31)(32)(33).Determination of the three-dimensional structures of Ras and GAP by X-ray crystallography represents an important milestone in the understanding of the catalysis mechanism. However, alterations of 0.01 Å in the GTP geometry, below the resolution of the X-ray structure analysis, induced by protein binding and charge shifts within GTP have been shown to contribute significantly to catalysis (16,19).…”

“…This is effected by GTPase-activating proteins (GAPs) that interact with Ras (6). The mechanisms of GTP hydrolysis have been extensively investigated both theoretically (7-22) and experimentally by X-ray crystallography (23-27), electron spin resonance (28-30), and FTIR spectroscopy (5,6,(31)(32)(33).…”

Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme

“…3 The results of time-resolved FTIR spectroscopy provided valuable information on the molecular reaction mechanism and dynamics in the corresponding protein systems. [4][5][6][7] Other kinetic measurements on the GTP hydrolysis reaction carried on Ras molecules complexed either with p120-GAP or with another GTPase accelerating protein neurofibromin (NF1) have been also reported. 5,[8][9][10][11][12] The basic conclusion from all these experimental studies can be formulated as follows: if the changes in GTP concentration in the chemical reactions can be measured then the observed rate constants at temperatures near 300K are close to 19 s -1 ; the observed rate constants for release of inorganic phosphate are about twice lower.…”