Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras

1997 ◽  
Vol 4 (9) ◽  
pp. 686-689 ◽  
Author(s):  
Mohammad Reza Ahmadian ◽  
Patricia Stege ◽  
Klaus Scheffzek ◽  
Alfred Wittinghofer
Keyword(s):  
Hydrolysis Reaction ◽  
Gtp Hydrolysis ◽  
Arginine Finger

scholarly journals GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

2000 ◽  
Vol 346 (2) ◽  
pp. 501-508 ◽  
Author(s):  
Zhimin LIANG ◽  
Timothy MATHER ◽  
Guangpu LI
Keyword(s):  
Biological Activity ◽  
Mutational Analysis ◽  
The Other ◽  
Hydrolysis Reaction ◽  
Rab Gtpase ◽  
Gtpase Activity ◽  
Phosphate Binding ◽  
Gtp Hydrolysis ◽  
Arginine Residues ◽  
Binding Loop

Structural and biochemical data indicate the importance of the phosphate-binding loop residues Gly12 and Gly13 of Ras both in the GTP hydrolysis reaction and in biological activity, but these two residues are not conserved in other Ras-related GTPases. To gain a better understanding of this region in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPase as a model for comparison, and substituted the Ala30 residue (the equivalent of Gly13 of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp contrast with the observation that a proline substitution at the neighbouring position (Gly12 of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution surprisingly stimulated the GTPase activity by 5-fold. Molecular modelling suggests that this built-in arginine mimics the catalytic arginine residues found in trimeric GTPases and GTPase-activating proteins in providing the positive charge to facilitate the GTP hydrolysis reaction. We investigated further the biological activity of the Rab5 mutants in relation to stimulating endocytosis. When expressed in cultured baby hamster kidney cells, both arginine and proline mutants, like wild-type Rab5, stimulated endocytosis. However, the arginine mutant was a more potent stimulator than the proline mutant (3-fold stimulation as against 1.7-fold). The tryptophan mutant, on the other hand, was completely deficient in activity in terms of the stimulation of endocytosis, demonstrating the importance of the phosphate-binding loop in Rab GTPase function.

scholarly journals Noncanonical Myo9b-RhoGAP Accelerates RhoA GTP Hydrolysis by a Dual-Arginine-Finger Mechanism

2016 ◽  
Vol 428 (15) ◽  
pp. 3043-3057 ◽  
Author(s):  
Fengshuang Yi ◽  
Ruirui Kong ◽  
Jinqi Ren ◽  
Li Zhu ◽  
Jizhong Lou ◽  
...  
Keyword(s):  
Gtp Hydrolysis ◽  
Arginine Finger

RanGAP mediates GTP hydrolysis without an arginine finger

Nature ◽  
2002 ◽  
Vol 415 (6872) ◽  
pp. 662-666 ◽  
Author(s):  
Michael J. Seewald ◽  
Carolin Körner ◽  
Alfred Wittinghofer ◽  
Ingrid R. Vetter
Keyword(s):  
Gtp Hydrolysis ◽  
Arginine Finger

scholarly journals Physicochemical Insights into Microscopic Events Driven by GTP Hydrolysis Reaction in Ras-GAP system

2021 ◽  
Author(s):  
Ikuo Kurisaki ◽  
Shigenori Tanaka
Keyword(s):  
Kinetic Energy ◽  
Chemical Species ◽  
Technical Difficulty ◽  
Coulomb Repulsion ◽  
Functional Expression ◽  
Hydrolysis Reaction ◽  
Dynamic Simulations ◽  
Gtp Hydrolysis ◽  
Product Systems ◽  
Functional Regions

Hydrolysis reaction of nucleotide triphosphate, ATP and GTP in particular, has been widely found as regulatory machinery for protein functional expression in the cell. Nonetheless, the microscopic mechanisms for functional regulations via the chemical reactions are mostly elusive so far, due to technical difficulty of both experimental observations and conventional theoretical simulations. We addressed the problem by examining the conjecture that Coulomb repulsion interaction between products, ADP/GDP and inorganic phosphate (Pi), execute the mechanical works upon the system. GTP hydrolysis reaction for Ras-GTP-GAP system was effectively simulated in the framework of classical atomistic molecular dynamic simulations by switching force field parameters between the reactant and product systems. We observed transient increase of kinetic energy of GDP and Pi, and the neighboring functional domains of Ras. One of such functional regions, P-loop, shows increase of nonbonded potential energy, which is retained even after gained kinetic energy is dissipated. This change is explained from rearrangement of hydrogen bonding between P-loop and GDP. Interestingly, even if increase of kinetic energy is suppressed, the above change is reproduced through GTP-GDP conversion. This observation suggests that conversion of chemical species itself plays essential roles in regulation rather than transient heat generation via the hydrolysis reactions.

scholarly journals Fourier Transform Infrared Spectroscopy on the Rap·RapGAP Reaction, GTPase Activation without an Arginine Finger

2004 ◽  
Vol 279 (44) ◽  
pp. 46226-46233 ◽  
Author(s):  
Partha P. Chakrabarti ◽  
Yan Suveyzdis ◽  
Alfred Wittinghofer ◽  
Klaus Gerwert
Keyword(s):  
Fourier Transform ◽  
Infrared Spectroscopy ◽  
Fourier Transform Infrared Spectroscopy ◽  
Structural Changes ◽  
Fourier Transform Infrared ◽  
Cleavage Reaction ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Gtpase Activation ◽  
Arginine Finger

GTPaseactivatingproteins (GAPs) down-regulate Ras-like proteins by stimulating their GTP hydrolysis, and a malfunction of this reaction leads to disease formation. In most cases, the molecular mechanism of activation involves stabilization of a catalytic Gln and insertion of a catalytic Arg into the active site by GAP. Rap1 neither possesses a Gln nor does its cognate Rap-GAP employ an Arg. Recently it was proposed that RapGAP provides a catalytic Asn, which substitutes for the Gln found in all other Ras-like proteins (Daumke, O., Weyand, M., Chakrabarti, P. P., Vetter, I. R., and Wittinghofer, A. (2004)Nature429, 197–201). Here, RapGAP-mediated activation has been investigated by time-resolved Fourier transform infrared spectroscopy. Although the intrinsic hydrolysis reactions of Rap and Ras are very similar, the GAP-catalyzed reaction shows unique features. RapGAP binding induces a GTP*conformation in which the three phosphate groups are oriented such that they are vibrationally coupled to each other, in contrast to what was seen in the intrinsic and the Ras·RasGAP reactions. However, the charge shift toward β-phosphate observed with RasGAP was also observed for RapGAP. A GDP·Piintermediate accumulates in the GAP-catalyzed reaction, because the release of Piis eight times slower than the cleavage reaction, and significant GTP synthesis from GDP·Piwas observed. Partial steps of the cleavage reaction are correlated with structural changes of protein side groups and backbone. Thus, the Rap·RapGAP catalytic machinery compensates for the absence of acis-Gln by atrans-Asn and for the catalytic Arg by inducing a different GTP conformation that is more prone to be attacked by a water molecule.

scholarly journals The Ability of GAP1IP4BP To Function as a Rap1 GTPase-Activating Protein (GAP) Requires Its Ras GAP-Related Domain and an Arginine Finger Rather than an Asparagine Thumb

2009 ◽  
Vol 29 (14) ◽  
pp. 3929-3940 ◽  
Author(s):  
Sabine Kupzig ◽  
Dalila Bouyoucef-Cherchalli ◽  
Sam Yarwood ◽  
Richard Sessions ◽  
Peter J. Cullen
Keyword(s):  
Binding Site ◽  
Molecular Model ◽  
Detectable Effect ◽  
Gtp Hydrolysis ◽  
Amino Terminal ◽  
Gtpase Activating Proteins ◽  
Close Proximity ◽  
Carboxy Terminal ◽  
Arginine Finger

ABSTRACT GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) that includes GAP1m, CAPRI, and RASAL. Composed of a central Ras GAP-related domain (RasGRD), surrounded by amino-terminal C2 domains and a carboxy-terminal PH/Btk domain, these proteins, with the notable exception of GAP1m, possess an unexpected arginine finger-dependent GAP activity on the Ras-related protein Rap1 (S. Kupzig, D. Deaconescu, D. Bouyoucef, S. A. Walker, Q. Liu, C. L. Polte, O. Daumke, T. Ishizaki, P. J. Lockyer, A. Wittinghofer, and P. J. Cullen, J. Biol. Chem. 281:9891-9900, 2006). Here, we have examined the mechanism through which GAP1IP4BP can function as a Rap1 GAP. We show that deletion of domains on either side of the RasGRD, while not affecting Ras GAP activity, do dramatically perturb Rap1 GAP activity. By utilizing GAP1IP4BP/GAP1m chimeras, we establish that although the C2 and PH/Btk domains are required to stabilize the RasGRD, it is this domain which contains the catalytic machinery required for Rap1 GAP activity. Finally, a key residue in Rap1-specific GAPs is a catalytic asparagine, the so-called asparagine thumb. By generating a molecular model describing the predicted Rap1-binding site in the RasGRD of GAP1IP4BP, we show that mutagenesis of individual asparagine or glutamine residues that lie in close proximity to the predicted binding site has no detectable effect on the in vivo Rap1 GAP activity of GAP1IP4BP. In contrast, we present evidence consistent with a model in which the RasGRD of GAP1IP4BP functions to stabilize the switch II region of Rap1, allowing stabilization of the transition state during GTP hydrolysis initiated by the arginine finger.

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