NDLI: The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins

Content Provider	Journal of Biological Chemistry (JBC)
Author	Zhang, Baolin Zhang, Yaqin Wang, Zhi-xin Zheng, Yi
Abstract	The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg2+ cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg2+ for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg2+ similarly, with dissociation constants in the submicromolar concentration. The presence of Mg2+, however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg2+, and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg2+, suggesting that one role of GEF is to displace bound Mg2+ from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg2+, indicating that the GEF-catalyzed nucleotide exchange involves a Mg2+-independent as well as a Mg2+-dependent mechanism. Although Mg2+ is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4–10-fold upon binding to Mg2+, andk cat values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg2+ was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg2+ cofactor. These studies directly demonstrate a role of Mg2+ in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg2+, whereas RhoGAP utilizes the Mg2+ cofactor to achieve high catalytic efficiency and specificity.
Related Links	http://www.jbc.org/content/275/33/25299.abstract
Ending Page	25307
Starting Page	25299
Page Count	9
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	33
Volume Number	275
DOI	10.1074/jbc.M001027200
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2000-08-18
Access Restriction	Open
Subject Keyword	Guanine nucleotide exchange factor (GEF) GTPase-activating protein (GAP) Glutathione S-transferase (GST) 2′(3′)-O-(N-methylanthraniloyl)GDP (mantGDP) 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) Guanosine 5′-3-O-(thio)triphosphate (GTPγS) ADP-ribosylation factor (ARF) Dithiothreitol (DTT) Β,γ-imidoguanosine 5′-triphosphate (GMP-PNP) MECHANISMS OF SIGNAL TRANSDUCTION
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Negative Regulation of Rho Family GTPases Cdc42 and Rac2 by Homodimer Formation

Characterization of a Fast Cycling ADP-ribosylation Factor 6 Mutant

Identification of Arfophilin, a Target Protein for GTP-bound Class II ADP-ribosylation Factors

Oligomerization of Rac1 GTPase Mediated by the Carboxyl-terminal Polybasic Domain

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac “THE EMPEROR'S NEW CLOTHES”

Guanine Nucleotide Exchange on ADP-ribosylation Factors Catalyzed by Cytohesin-1 and Its Sec7 Domain

αPix Stimulates p21-activated Kinase Activity through Exchange Factor-dependent and -independent Mechanisms

A Novel Binding Protein Composed of Homophilic Tetramer Exhibits Unique Properties for the Small GTPase Rab5

Ran-dependent Signal-mediated Nuclear Import Does Not Require GTP Hydrolysis by Ran

The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins

Similar Documents

Negative Regulation of Rho Family GTPases Cdc42 and Rac2 by Homodimer Formation

Characterization of a Fast Cycling ADP-ribosylation Factor 6 Mutant

Identification of Arfophilin, a Target Protein for GTP-bound Class II ADP-ribosylation Factors

Oligomerization of Rac1 GTPase Mediated by the Carboxyl-terminal Polybasic Domain

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac “THE EMPEROR'S NEW CLOTHES”

Guanine Nucleotide Exchange on ADP-ribosylation Factors Catalyzed by Cytohesin-1 and Its Sec7 Domain

αPix Stimulates p21-activated Kinase Activity through Exchange Factor-dependent and -independent Mechanisms

A Novel Binding Protein Composed of Homophilic Tetramer Exhibits Unique Properties for the Small GTPase Rab5

Ran-dependent Signal-mediated Nuclear Import Does Not Require GTP Hydrolysis by Ran

The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins