Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time

Abstract

The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P(3)-1-(2-nitro)phenylethyl guanosine 5'-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP x Ras x GDP x AlF(x) transition state analog is known, which represents a "snap shot" along the reaction-pathway. As now revealed, binding of GAP to Ras x GTP shifts negative charge from the gamma to beta phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP x Ras x GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between beta and gamma phosphate is cleaved. In the intermediate, the released P(i) is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P(i) from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein-protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.

Figure 1

The absorbance changes in the infrared between 1800 and 950 cm⁻¹ during the intrinsic GTPase reaction of Ras and during the GAP-catalyzed reaction are shown. The absorbance changes of the intrinsic reaction can be described by a single exponential function as seen for example at 1143 cm⁻¹ for GTP and 1104 cm⁻¹ for GDP. In the GAP-catalyzed reaction, an intermediate accumulates as seen for example at 1143 cm⁻¹. The fitted curves of the global multiexponential kinetic analysis are shown (note the difference in log time scale). Absorbance changes at single wave numbers as function of time are shown in Fig. 4.

Figure 2

(a) The amplitude spectra of k₂, which describe the A to B transition are shown (black lines). Negative bands belong to the GTP state and positive bands to the intermediate. In addition, the amplitude spectra of the labeled compounds are shown (colored lines). The ¹⁸O-labeled positions in GTP are indicated by colored oxygens. (b) Differences between the amplitude spectra of labeled and unlabeled GTP as shown in a are presented to visualize the frequency shifts. The shifts of labeled bands cause difference bands, illustrated by shaded areas. The original frequency is given in black and the shifted position in color. This is available enlarged as Figs. 7 and 8, which are published as supplemental data on the PNAS web site, www.pnas.org.

Figure 3

(a) The amplitude spectrum of k₃ (black lines), which describes the transition from the intermediate to the final GDP state. Negative bands now belong to the intermediate and positive bands to the GDP state. In addition, the amplitude spectra of the labeled compounds are shown (colored lines) as in Fig. 2a. (b) Differences between the amplitude spectra of labeled and unlabeled GTP as shown in a, presented as in Fig. 2a. This is available enlarged as Figs. 9 and 10, which are published as supplemental data.

Figure 4

Typical absorbance changes of the GAP-catalyzed reaction are shown. Besides the data (crosses), the fitted curves (solid line) and the individual contributions (k_i) of the multi-exponential fit analysis are shown. In contrast to the intrinsic Ras-catalyzed reaction, which can be described with one exponential function and the rate constant k = (5.1 ± 0.5) × 10⁻⁴ s⁻¹ at 310 K, the absorbance changes have to be described with at least three rate constants k₁ = (6.8 ± 4.1) s⁻¹, k₂ = (0.77 ± 0.24) s⁻¹, and k₃ = (0.102 ± 0.027) s⁻¹ at 260 K. k₁ represents the photolysis of caged GTP and the appearance of released γ phosphate as indicated at 1143 cm⁻¹. k₂ shows the cleavage of γ phosphate again seen at 1143 cm⁻¹ and the appearance of protein-bound P_i at 1113 cm⁻¹. k₃ shows the decay of the protein bound P_i at 1113 cm⁻¹ and its release from the protein–protein complex into the bulk medium at 1078 cm⁻¹. k₃ represents the rate-limiting step. It is clearly seen that GAP catalyses the bond cleavage reaction.

Figure 5

The frequency shifts of GTP, GDP, and P_i vibrations induced by different binding partners are listed (–16, 22).

Figure 6

The cartoon summarizes the results: the shift of negative charges from γ-GTP to β oxygens because of Ras binding and the additional larger shift mostly to the (S_P)-β oxygen because of GAP binding by Arg-789 and/or Lys-16 and backbone NH groups of the highly conserved P-loop are illustrated. Mg²⁺, Lys, and Arg are considered to be the key charged residues holding like “molecular tweezers” charges on β oxygens. This charge shift reduces the free activation energy for β–γ bond cleavage in the A to B transition with k₂ (compare Fig. 4, 1143 cm⁻¹). The P_i is strongly bound, most likely to GAP, in the intermediate B. P_i release is rate limiting, with k₃ in the B to C transition (compare Fig. 4, 1113, 1078, 1104 cm⁻¹). The structural arrangement is based on structural models of refs. , , and .