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

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 ␥-phosphate, there appears a H2PO 4 proteins ͉ reaction mechanism ͉ FTIR spectroscopy ͉ signal transduction T he guanine nucleotide-binding protein Ras regulates several signal transduction processes involved in cell growth and differentiation (1). Ras serves as a prototype for the superfamily of GTPases that cycle between the active GTPbound state and the inactive GDP-bound state. The switchingoff of signal transduction is accomplished by GTP hydrolysis, which is a phosphoryl transfer from GTP to water. This process is slow for Ras⅐GTP, allowing further control of this process by GTPase-activating proteins (GAPs), which stimulate the reaction by several orders of magnitude (2). The Ras protein has been extensively studied by various methods, including x-ray crystallography (3-5), NMR (6), computation (7-10), and FTIR spectroscopy (11)(12)(13)(14)(15). For the slow intrinsic reaction, a substrate-assisted mechanism has been inferred from biochemical and computational studies, whereby ␥-phosphate acts as a general base to activate the nucleophilic water (16,17). Accumulating intermediates in GAP-catalyzed reactions have recently been observed for both the Ras⅐RasGAP and the Rap⅐RapGAP reaction, and these intermediates can either decompose to the products GDP and P i or regenerate GTP (11,18,19). Recently, a crystal structure of an intermediate of an enzyme-catalyzed phosphoryl transfer reaction was reported (20). This intermediate was analyzed as a pentacovalent phosphate structure, but there is considerable controversy concerning this interpretation (21-23).Time-resolved FTIR (trFTIR) difference spectroscopy monitors protein reactions at the atomic level in real time (24,25). In the present application, Ras in the presence of saturating amounts of NF1-333, the catalytic GAP domain of neurofibromin, was loaded with caged GTP, which cannot be hydrolyzed by the Ras⅐RasGAP system. Ras⅐GTP was generated by a laser f lash, and the subsequent hydrolysis reaction was monitored by tr-FTIR with a time resolution of milliseconds. The phosphate vibrations were assigned by using 18 Olabeled caged GTP (12). A global multiexponential kinetic analysis of the trFTIR data obtained at 260 K was performed by using a sum of three exponential functions for the GAPcatalyzed GTPase reaction of Ras (11).The first process, with the rate constant k 1 (data not shown), describes the appearance of GTP from the precursor caged GTP (11). At the second rate (rate constant k 2 ), GTP disappears, and an intermediate appears that is assigned to protein-bound P i , initially without implication for its structure. P i is released from the protein in the rate-limiting step, which is described by the third ra...

The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steadystate kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1 IP4BP and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-a6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.

Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP-to-GTP exchange. This modifies the Ras surface (Ras(on)GTP) and enables effector binding, which then activates signal-transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to "off" (Ras(off)GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras(off)-to-Ras(on) transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time-resolved FTIR (trFTIR) spectroscopy. In the transition from Ras(off) to Ras(on) a GTP-bound intermediate is now identified, in which effector binding is still prevented (Ras(off)GTP). The loop movement from Ras(off)GTP to Ras(on)GTP was directly monitored by the C=O vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s(-1) at 260 K. A small molecule that shifted the equilibrium from the Ras(on)GTP state towards the Ras(off)GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.

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