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Review Ras-Catalyzed Hydrolysis of GTP: a New Perspective from Model Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8160-8166, August 1996 Review Ras-catalyzed hydrolysis of GTP: A new perspective from model studies Karen A. Maegley, Suzanne J. Admiraal, and Daniel Herschlag* Department of Biochemistry, B400 Beckman Center, Stanford University, Stanford, CA 94305-5307 Communicated by James A Spudich, Stanford University Medical Center, Stanford, CA, March 6, 1996 (received for review December 21, 1995) ABSTRACT Despite the biological and medical impor- A new catalytic mechanism is then proposed, involving a tance of signal transduction via Ras proteins and despite hydrogen bond to the ,3-y bridge oxygen of GTP. This proposal considerable kinetic and structural studies of wild-type and is consistent with pre-existing structural, spectral, and ener- mutant Ras proteins, the mechanism of Ras-catalyzed GTP getic data. hydrolysis remains controversial. We take a different ap- proach to this problem: the uncatalyzed hydrolysis of GTP is Background: The Nature of the Transition State analyzed, and the understanding derived is applied to the for GTP Hydrolysis Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton There is a continuum of potential transition state structures for abstraction from the attacking water by a general base and phosphoryl transfer reactions, ranging from dissociative to stabilization of charge development on the y-phosphoryl associative depending on the nature of the bonding (i.e., the oxygen atoms would not be catalytic. Rather, this analysis electronic distribution; Scheme I). focuses attention on the GDP leaving group, including the 1-y bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in Dissociative which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strength- ened transition state interactions of the active site lysine, H 1 Lys-16, with the f3-nonbridging phosphoryl oxygens and a bO+ o v -OR I-R network of interactions that positions the nucleophilic water molecule and y-phosphoryl group with respect to one another (-2) may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the 18-y bridge oxygen with an Arg side chain that is provided in trans by GAP. -s(-24-c)iv The conclusions for Ras and related G proteins are expected to Associative apply more widely to other enzymes that catalyze phosphoryl Scheme I (-PO2-) transfer, including kinases and phosphatases. A dissociative transition state is dominated by bond cleavage; Ras and other G proteins are active in signaling when GTP is in the extreme case, the bond to the outgoing leaving group is bound. Hydrolysis of GTP to give bound GDP turns these fully or nearly broken (Scheme I top, large 8-), and the bond signaling proteins off (1-3). For this reason, the mechanism of to the incoming nucleophile is absent or barely formed (small GTP hydrolysis by Ras and other G proteins has elicited much 8+). To maintain conservation of charge, there must then be interest. Nevertheless, despite a wealth of structural, kinetic, a loss of charge on the phosghoryl group (i.e., the - P03 group) and site-directed mutagenesis data, the catalytic mechanism of being transferred. In contrast, in an associative transition state Ras remains controversial. there is a large amount of bond formation to the incoming Crucial to an understanding of enzymatic catalysis is knowl- nucleophile (Scheme I bottom, large 8+) but only a small edge of the nature of the transition state of the reaction and amount of bond cleavage to the outgoing leaving group (small how that transition state differs from the ground state; tran- 8-); thus, there is a gain in charge on the phosphoryl group sition state theory defines catalysis as stabilization of a reac- being transferred. A wealth of physical organic data obtained tion's transition state relative to its ground state. An under- over the past 40 years has implicated a dissociative, metaphos- lying feature comrmon among the several distinct models phate-like transition state in reactions of phosphate mono- presented for Ras catalysis is an explicit or implicit assumption esters, acyl phosphates, and phosphorylated amines (5-10, t). that the transition state is associative in character. However, recent model studies have shown that the transition state for Abbreviations: GTP[,yS], guanosine 5'-[,y-thio]triphosphate; GMP- GTP hydrolysis in solution is dissociative rather than associa- PNP, guanylyl f3,y-imidodiphosphate; GMPPCP, guanylyl 13,y- tive (4). This transition state information is used to evaluate methylenediphosphonate. previously proposed mechanisms from a chemical perspective. *e-mail: [email protected]. tA transition state can be dissociative or metaphosphate-like in nature and yet occur in a second-order reaction that has some amount of The publication costs of this article were defrayed in part by page charge association with an incoming nucleophile in the transition state. Thus, payment. This article must therefore be hereby marked "advertisement" in the absence of racemization of stereochemistry of phosphoryl groups accordance with 18 U.S.C. §1734 solely to indicate this fact. in nonenzymatic and enzymatic reactions, although providing evi- 8160 Downloaded by guest on September 28, 2021 Review: Maegley et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8161 Recent linear free energy relationships have shown that -0.14 transfer of the y-phosphoryl group of GTP proceeds via a +0.26 dissociative, metaphosphate-like transition state, analogous to the previously characterized transition states for reactions of other monosubstituted phosphoryl compounds (4). It has been H ~~/\ suggested that metal ion coordination of the y-phosphoryl H -- oxygens in GTP-Mg could increase the susceptibility of the P GMP phosphorus to nucleophilic attack and therefore increase the +0.05 X ---... of the transition state However, associative character (15-21). -0.55 comparisons of linear free energy relationships for reactions of +0.26 GTP and GTP-Mg strongly suggest that Mg2+ does not +0.26 increase the associative character of the transition state (ref. 4; -0.14 see also refs. 22 and 23). Furthermore, large changes in reactivity induce only small changes in the nature of the Change in charge: transition state for phosphoryl transfer, suggesting that these ground to transition state transition states are difficult to change (23-25). Nevertheless, could the greater preponderance of positively FIG. 1. Changes in charge in the nonenzymatic hydrolysis of charged side chains and metal ions in an enzymatic active site GTP4-. The transition state geometry is shown, with color intensity change the nature of the transition state from dissociative to proportional to the charge development in the transition state relative associative? This proposal was recently tested with Escherichia to the ground state. The dark blue color of the 13-y bridge oxygen coli alkaline phosphatase, which utilizes two active site zinc indicates that it undergoes the largest change in charge. The crude ions and an Arg side chain. The large dependence of rate on quantitative estimates were derived from linear free energy relation- leaving group pKa suggested a large amount of bond cleavage ships (4) and are used solely for qualitative arguments in the text. This in a dissociative transition state (26). Heavy atom isotope figure was adapted from ref. 4. effects similarly suggest a dissociative transition state for transition state (4), the pKat for loss of a proton from the reactions of two protein phosphatases (27). water is to be --14 in the transition In summary, the simplest expectation is that the transition nucleophilic expected state for GTP hydrolysis at the active site of Ras is dissociative in character. Although not proven, this chemical perspective provides a logical starting point for a discussion of enzymatic catalysis. Any proposal of a predominantly associative transi- tion state for phosphoryl transfer requires a concomitant proposal that some distinct feature of the enzyme changes the transition state. Reaction 1 Previous Mechanistic Proposals for the Ras GTPase Reaction state.§ The pKat of 14 is much higher than the pKa -2 for deprotonation of the acid form of the Gln side chain carbonyl The change in charge distribution that occurs in proceeding [i.e., Gln(C==OH+)NH2] (33). from the ground state to the transition state for GTP hydro- lysis is shown in Fig. 1. These changes are used in this section to analyze proposed mechanisms of catalysis by Ras and other This means that the hypothetical equilibrium of Reaction 1 lies G proteins. The purpose is not to survey all previously very far to the left (Kestimated 10-16) so that no significant proposed mechanisms but rather to discuss the most common proton transfer from the attacking water to Gln-61 is expected of these from a chemical perspective. in a dissociative transition state (see also ref. 34). General Base Catalysis? It has been stated recently that: Replacement of Gln-61 by Glu increases the rate of GTP "The major unresolved problem [of Ras catalysis] is the nature hydrolysis by 20-fold, and this result has been considered of the general base activating the catalytic water molecule evidence that Gln-61 acts as a general base (35). However, Glu which acts as the nucleophile in the hydrolysis reaction" (28). would not be expected to abstract a proton in the transition However, in a dissociative transition state, there is little bond state for the same reasons outlined above for Gln. Perhaps the formation to the incoming water nucleophile and therefore migration of negative charge away from the attacking water little charge development (Scheme I and Fig.
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