γ-Phosphate Protonation and pH-dependent Unfolding of the Ras·GTP·Mg2+ Complex

Abstract

The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis. Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the ␥-phosphate of GTP could be the general base. The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras⅐GTP⅐Mg 2؉ and Ras⅐GDP⅐Mg 2؉ complexes. Isotopeedited IR studies of the Ras⅐GTP⅐Mg 2؉ complex show that GTP remains bound to Ras at pH as low as 2.0 and that the ␥-phosphate is not protonated at pH > 3.3, indicating that the active site decreases the ␥-phosphate pK a by at least 1.1 pK a units compared with solution. Amide I studies show that the Ras⅐GTP⅐Mg 2؉ and Ras⅐GDP⅐Mg 2؉ complexes partially unfold in what appear to be two transitions. The first occurs in the pH range 5.4-2.6 and is readily reversible. Differences in the pH-unfolding midpoints for the Ras⅐GTP⅐Mg 2؉ and Ras⅐GDP⅐Mg 2؉ complexes (3.7 and 4.8, respectively) reveal that the enzyme-␥-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent unfolding of the Ras⅐GTP⅐Mg 2؉ complex provides an alternative interpretation of the data that had been used to support the ␥-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.The cellular activities of the Ras protein are causally linked to tumorogenesis in a high percentage and wide variety of human tissues (1). Binding of this cellular messenger to Raf-1 protein kinase initiates a metabolic cascade that leads to cell growth and differentiation (2, 3). Ras plays essential roles in regulating the cell cycle (4, 5), apoptosis (6), mitogen-activated protein kinase pathways (7,8), and retroviral activation (9). The allosteric interactions that mediate signaling between GTPases and their targets are typically switched from on to off by the hydrolysis of GTP (10, 11); however, there are exceptions (12). The interdependence of hydrolysis and signaling functions of GTPases has fostered intensive studies of the chemical mechanism of the Ras-catalyzed hydrolysis of GTP.Classical general base theory suggests that Ras might catalyze hydrolysis by abstracting a proton from the nucleophilic water, thereby activating it to attack the ␥-phosphate. Careful inspection of the GTP-bound active site of Ras suggests five possible general base candidates (Asp-38, Asp-57, Gln-61, Glu-62, and glu-63). The GTPase activity of mutants of each of these amino acids has been studied (13-16), and it is generally agreed that the presumed base has not been identified. The lack of a conclusive identification of the base has spawned alternative suggestions for the chemical mechanism. A recent theory that has attracted considerable at...

Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate

“…The shift in the FTIR spectra in going from GTP in solution to GTP on RAS is small, and this was taken as support for a dianionic state for the ␥-phosphate on GTP-bound RAS at pH 7.5 (24). Once this assumption was accepted, a deprotonated GTP became the starting point for quantum mechanical/classical mechanical (QM/MM) calculations and was used in the interpretation of subsequent FTIR spectra of GTP on RAS.…”

“…All proposed mechanisms to date assume that the GTP phosphate groups are fully deprotonated on the enzyme (total charge of Ϫ4), as they are expected to be in solution at physiological pH (7.0 -7.2) in the presence of magnesium (24). Detecting a possible change in pK a of any of the GTP phosphate groups on the enzyme is a challenge due to the fact that hydrogen atoms in biological systems are difficult to visualize even in high resolution x-ray crystal structures.…”

Two sides of the same coin: How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase

“…Band shifts were observed with respect to the spectrum of free ATP, however, the overall conclusion was that the phosphate groups experience interactions of similar strength in the protein environment and in water. An interesting difference to similar studies on GTP bound to Ras [1,22,24,28] is that the vibrations of α-, β-and γ -phosphate are largely decoupled in this case, whereas they are coupled in the ATPase ATP complex.…”

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“…Heterogroups (nucleotides, cofactors) were included in the protonation state that had been manually assigned. A full deprotonation of the phosphate groups was assigned as this was shown to be the case in the literature ( 38 , 39 ). For G α i1 -Mg 2+ -GDP simulations, a Mg 2+ ion with four bound water molecules was placed next to β -GDP.…”