Understanding the Catalytic Mechanism of GTPase-Activating Proteins:  Demonstration of the Importance of Switch Domain Stabilization in the Stimulation of GTP Hydrolysis†

Biochemistry ◽  
2002 ◽  
Vol 41 (52) ◽  
pp. 15644-15653 ◽  
Author(s):  
Nancy J. Fidyk ◽  
Richard A. Cerione
Keyword(s):  
Catalytic Mechanism ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Domain Stabilization ◽  
Stimulation Of

scholarly journals Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP–ASAP1 complex

2007 ◽  
Vol 402 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Ruibai Luo ◽  
Bijan Ahvazi ◽  
Diana Amariei ◽  
Deborah Shroder ◽  
Beatriz Burrola ◽  
...  
Keyword(s):  
Binary Complex ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Steady State Kinetics ◽  
Catalytically Active ◽  
Ras Family ◽  
Src Homology ◽  
Significant Conformational Change

Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a kcat of 57±5 s−1 and a Km of 2.2±0.5 μM determined by steady-state kinetics and a kcat of 56±7 s−1 determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4−), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than Km. Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the kcat. Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP–ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

Life Sciences ◽  
1982 ◽  
Vol 30 (3) ◽  
pp. 269-275 ◽  
Author(s):  
Klaus Aktories ◽  
Günter Schultz ◽  
Karl H. Jakobs
Keyword(s):  
Adenosine Receptor ◽  
Gtp Hydrolysis ◽  
Stimulation Of

scholarly journals Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

2009 ◽  
Vol 20 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Lena Kliouchnikov ◽  
Joëlle Bigay ◽  
Bruno Mesmin ◽  
Anna Parnis ◽  
Moran Rawet ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
In Vitro Assays ◽  
Minor Role ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Golgi Localization ◽  
Adp Ribosylation Factor ◽  
Fusion Approach

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.

scholarly journals Stress-dependent inhibition of cell polarity through unbalancing the GEF/GAP regulation of Cdc42

2021 ◽  
Author(s):  
Clàudia Salat-Canela ◽  
Mercè Carmona ◽  
Rebeca Martín-García ◽  
Pilar Pérez ◽  
José Ayté ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Dependent Inhibition ◽  
Stress Dependent

Cdc42 rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and by Guanine-nucleotide Exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its down-regulation. We describe here that oxidative stress causes Sty1 kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at poles and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles by either eliminating two Cdc42 GAPs or through the constitutive tethering of a GEF to the cell tips, and biochemically demonstrate that Rga3 is a direct substrate of Sty1. We propose that stress-activated Sty1 promotes GTP hydrolysis and prevents GEF activity at the cell tips, thus leading to the inhibition of Cdc42 and polarized growth cessation.

scholarly journals Structural insights into TSC complex assembly and GAP activity on Rheb

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Huirong Yang ◽  
Zishuo Yu ◽  
Xizi Chen ◽  
Jiabei Li ◽  
Ningning Li ◽  
...  
Keyword(s):  
Tuberous Sclerosis ◽  
Catalytic Mechanism ◽  
Coiled Coil ◽  
Small Gtpase ◽  
Dimerization Domain ◽  
Gtp Hydrolysis ◽  
Complex Assembly ◽  
Complex Functions ◽  
Biochemical Analyses ◽  
Hydrolysis Of

AbstractTuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.

scholarly journals Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations

2012 ◽  
Vol 109 (38) ◽  
pp. 15295-15300 ◽  
Author(s):  
Till Rudack ◽  
Fei Xia ◽  
Jürgen Schlitter ◽  
Carsten Kötting ◽  
Klaus Gerwert
Keyword(s):  
Nucleophilic Attack ◽  
Nonbridging Oxygen ◽  
Difference Spectra ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
X Ray ◽  
Cellular Processes ◽  
Gtpase Activating Proteins ◽  
Biomolecular Simulations ◽  
Oxygen Atoms

Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and Pi by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 105 compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 105 compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.

What vibrations tell us about GTPases

2015 ◽  
Vol 396 (2) ◽  
pp. 131-144 ◽  
Author(s):  
Carsten Kötting ◽  
Klaus Gerwert
Keyword(s):  
Small Gtpases ◽  
Small Gtpase ◽  
Attenuated Total Reflection ◽  
Transport Mechanisms ◽  
Spatiotemporal Resolution ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Gtpase Activating Proteins ◽  
Catalytic Mechanisms ◽  
Insight Into

Abstract In this review, we discuss how time-resolved Fourier transform infrared (FTIR) spectroscopy is used to understand how GTP hydrolysis is catalyzed by small GTPases and their cognate GTPase-activating proteins (GAPs). By interaction with small GTPases, GAPs regulate important signal transduction pathways and transport mechanisms in cells. The GTPase reaction terminates signaling and controls transport. Dysfunctions of GTP hydrolysis in these proteins are linked to serious diseases including cancer. Using FTIR, we resolved both the intrinsic and GAP-catalyzed GTPase reaction of the small GTPase Ras with high spatiotemporal resolution and atomic detail. This provided detailed insight into the order of events and how the active site is completed for catalysis. Comparisons of Ras with other small GTPases revealed conservation and variation in the catalytic mechanisms. The approach was extended to more nearly physiological conditions at a membrane. Interactions of membrane-anchored GTPases and their extraction from the membrane are studied using the attenuated total reflection (ATR) technique.

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