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   Gln^cat in Ras) ([105]8, [106]42), indicating that Gln^catis not
   required for binding of W^nuc in the ground state. It has been pointed
   out that the basicity of glutamine is low, and it is therefore unlikely
   that Gln^cat acts as the general base that deprotonates W^nuc
   ([107]43). Rather, an oxygen of the presumably dianionic γ-phosphate is
   proposed to serve this function in Ras ([108]30, [109]31). Hence,
   Gln^cat may only polarize W^nuc in the ground state.

   X-ray crystallographic studies have indicated that Gln^catstabilizes
   the transition state ([110]2), as originally proposed by Priveet al.
   ([111]43). Structures of G[iα1] and G[tα] complexed with the transition
   state analog GDP·AlF[4] ^−, reveal Gln^catpositioned within the active
   site and directly interacting with a fluorine substituent and W^nuc of
   the GDP·AlF[4] ^−·W^nuc complex. Fig.[112]2 C shows these interactions
   in the RGS4·G[iα1]·GDP·AlF[4] ^−complex ([113]44). It was proposed that
   the amino group of Gln^cat stabilizes negative character on the
   equatorial oxygen of the transition state and its carbamoyl oxygen
   stabilizes the attacking nucleophilic water. A similar configuration is
   observed in the Ras-GAP·Ras·GDP·AlF[4] ^− and the
   Rho-GAP·Cdc42Hs·GDP·AlF4^− complexes ([114]45-47). In addition,
   mutations that perturb the transition state conformation of Gln^cat
   abolish GTPase activity ([115]42, [116]48). These observations indicate
   that Gln^cat stabilizes the transition state for GTP hydrolysis.

   The GppNHp complex provides novel insights into both the mechanism of
   GTP hydrolysis as well as to the role of both Arg^cat and Gln^cat in
   the ground state E·S complex. GppNHp, but not GTPγS, permits a water
   molecule, W^600, to occupy a position in which it could act as the
   ultimate proton acceptor from W^nuc. Water molecules in similar, but
   not identical, positions are present in the GppNHp- or GppCp-bound
   complexes of Ras and Rac1 ([117]32, [118]40). A proton could be relayed
   from W^nuc to W^600 via O1G of the GTP γ-phosphate. This substituent
   does not otherwise participate in hydrogen bonds with the protein and
   corresponds to the thiol of GTPγS. The basicity of W^600may be enhanced
   by hydrogen bond formation with Glu^43, which is well conserved in Gα
   proteins with the exception of G[zα] where it is replaced with an
   asparagine residue. Glu^43 also forms a hydrogen-bonded ion pair with
   Arg^178. In this conformation, Arg^178 is restrained from interacting
   with the γ-phosphate of GTP. Transfer of a proton from W^nuc to W^600
   would tend to weaken this ion pair, releasing Arg^178 to stabilize the
   incipient pentacoordinate phosphoryl transition state. W^600 also
   blocks the side chain of Gln^204 from interacting with the
   pentacoordinate phosphate. Thus, until it diffuses from the active
   site, W^600 impedes the reorganization of the catalytic site that is
   required for transition state stabilization.

   Gln^204 is anchored in a noncatalytic conformation by hydrogen bonds to
   both W^nuc and Ser^206(Ser^206 is substituted by an Asp in G[αs],). In
   the ground state, Gln^204 could orient and perhaps activate W^nuc;
   however, to stabilize the transition state as represented by G protein
   GDP·AlF[4] ^−complexes, Gln^204 must sever its hydrogen bond with
   Ser^206 and W^nuc and rotate ≈120° about χ[1] and ≈90° about χ[2] and
   χ[3] (to gauche+ and gauche−, respectively) such that its carbamoyl
   group donates a hydrogen bond to the equatorial oxygen of the
   pentacoordinate γ-phosphoryl group and accepts a hydrogen bond from
   W^nuc. Such would incur a substantial penalty in catalytic efficiency
   and perhaps account, at least in part, for the low catalytic rate of
   GTP hydrolysis in G[α] and perhaps in other G proteins.

   We propose that the ground state G[iα1]·GTP complex is “auto-inhibited”
   with Gln^cat locked into an unproductive conformation. Active site
   residues in the EF-Tu·GppNHp complex also assumes anti-catalytic
   positions; in this case His^cat, the residue corresponding to Gln^cat,
   cannot interact with the substrates because of steric interference by
   other active site residues ([119]41). In G[iα1], catalysis could occur
   only if the bonds that hold Gln^cat in this position are broken, and
   the side chain freed to interact with the pentacoordinate transition
   state. This model predicts that changes that disrupt the ground state
   conformation of Gln^204, while not otherwise compromising the active
   site, would increasek [cat].

   RGS proteins, which accelerate the rate of GTP hydrolysis by G[iα1] by
   50–100-fold, may act in part by destabilizing the ground state
   conformation of Gln^cat, as well as stabilizing its productive
   conformation in the transition state ([120]44). The crystal structure
   of the RGS4·G[iα1]·GDP·AlF[4] ^−complex demonstrates that RGS4
   stabilizes the active site of G[iα1] in the conformation corresponding
   to that of the transition state complex (Fig. [121]2 C) ([122]44). No
   residues from RGS4 are inserted into the active site except Asn^128,
   which could enhance catalysis by aiding in binding, orienting, and
   polarizing W^nuc in the pre-transition state complex ([123]44).

   However, superposition of G[iα1]·GppNHp and G[iα1]·GDP·AlF[4] ^− from
   the RGS4·G[iα1]·GDP·AlF[4] ^−complex reveals that the carbamoyl groups
   of Gln^204 and Asn^128 occupy nearly the same positions, although the
   side chains approach from opposite directions (Fig. [124]2 D). Further,
   both residues are positioned such that they can bind the nucleophilic
   water and Ser^206. We suggest that Asn^128 of RGS4 displaces the side
   chain of Gln^204 from its “anti-catalytic” position in the ground
   state, freeing it to participate in stabilization of the transition
   state.

   Mutational analysis of RGS proteins supports this hypothesis. Mutation
   of Asn^131 in hRGSr (analogous to Asn^128 of RGS4) to either serine or
   glutamine resulted in a relatively small decrease in the k [cat] of
   G[tα] ([125]49). In addition, hRGSr in which Asn^131 was mutated to
   leucine or alanine also retains substantial stimulatory activity, and
   the loss of activity that was observed could be attributed to weakened
   binding of these mutants to G[tα]. Similar mutagenic studies have been
   performed with RGS4 ([126]50). Mutants of Asn^128analogous to those of
   hRGSr Asn^131 were modeled in the structure of the “RGS4·G[iα1]·GppNHp”
   complex. In all cases these residues were in steric conflict with
   Gln^204. These findings indicate that the bulk and binding of the
   residue at position 128 is important to the stimulatory activity of
   RGS4 although it is unlikely that it has a direct catalytic role in
   stimulation of GTPase activity ([127]49).

   The evidence presented is consistent with a self-inhibited or
   anti-catalytic model of the ground state of G[α] proteins, and a role
   for RGS proteins in stimulating GTPase activity by releasing G[α]
   subunits from this ground state while stabilizing the transition state.
   [128]Previous Section[129]Next Section

 §2§ Footnotes §2§

     * [130]↵* The costs of publication of this article were defrayed in
       part by the payment of page charges. The article must therefore be
       hereby marked “advertisement” in accordance with 18 U.S.C. Section
       1734 solely to indicate this fact.
       The atomic coordinates and structure factors (code1cip) has been
       deposited in the Protein Data Bank, Brookhaven National Laboratory,