Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogs

ABSTRACT The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coliand purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90° light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (∼10 min) than E. coliFtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg2+and GTP. The minimum concentration of FtsZ needed for polymerization is 3 μM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

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Involvement of Guanosine Triphosphate (GTP) Hydrolysis in the Mechanism of Tubulin Polymerization: Regulation of Microtubule Dynamics at Steady State by a GTP Cap

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1986.tb38427.x ◽

1986 ◽

Vol 466 (1 Dynamic Aspec) ◽

pp. 496-509 ◽

Cited By ~ 17

Author(s):

D. PANTALONI ◽

M-F. CARLIER

Keyword(s):

Steady State ◽

Microtubule Dynamics ◽

Tubulin Polymerization ◽

Guanosine Triphosphate ◽

Gtp Hydrolysis

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Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, from Thermotoga maritima

Journal of Bacteriology ◽

10.1128/jb.182.24.7078-7082.2000 ◽

2000 ◽

Vol 182 (24) ◽

pp. 7078-7082 ◽

Cited By ~ 40

Author(s):

Kunitoshi Yamanaka ◽

Jihwan Hwang ◽

Masayori Inouye

Keyword(s):

Thermotoga Maritima ◽

Binding Protein ◽

Protein A ◽

Hydrolysis Rate ◽

Gtpase Activity ◽

Gtp Binding Protein ◽

Gene Encoding ◽

Gtp Hydrolysis ◽

Gtp Binding

ABSTRACT A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium.T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. Km andk cat at 70°C were 833 μM and 9.3 min−1, respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.

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Biochemical Characterization of Recombinant Hepatitis C Virus Nonstructural Protein 4B: Evidence for ATP/GTP Hydrolysis and Adenylate Kinase Activity†

Biochemistry ◽

10.1021/bi801747p ◽

2009 ◽

Vol 48 (5) ◽

pp. 906-916 ◽

Cited By ~ 40

Author(s):

Aaron A. Thompson ◽

Aihua Zou ◽

Jiangli Yan ◽

Rohit Duggal ◽

Weidong Hao ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Adenylate Kinase ◽

Kinase Activity ◽

Biochemical Characterization ◽

Nonstructural Protein ◽

Recombinant Hepatitis ◽

Gtp Hydrolysis

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Characterization of a GTPase-activating Protein That Stimulates GTP Hydrolysis by Both ADP-ribosylation Factor (ARF) and ARF-like Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.271.39.24005 ◽

1996 ◽

Vol 271 (39) ◽

pp. 24005-24009 ◽

Cited By ~ 25

Author(s):

Min Ding ◽

Nicolas Vitale ◽

Su-Chen Tsai ◽

Ronald Adamik ◽

Joel Moss ◽

...

Keyword(s):

Gtpase Activating Protein ◽

Gtp Hydrolysis ◽

Adp Ribosylation ◽

Adp Ribosylation Factor

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Arl2 and Arl3 Regulate Different Microtubule-dependent Processes

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0929 ◽

2006 ◽

Vol 17 (5) ◽

pp. 2476-2487 ◽

Cited By ~ 112

Author(s):

Chengjing Zhou ◽

Leslie Cunningham ◽

Adam I. Marcus ◽

Yawei Li ◽

Richard A. Kahn

Keyword(s):

Common Ancestor ◽

Tubulin Polymerization ◽

Mitotic Spindles ◽

Eukaryotic Evolution ◽

Cycle Arrest ◽

Initial Characterization ◽

M Phase ◽

Functional Features ◽

Dependent Processes

Arl2 and Arl3 are closely related members of the Arf family of regulatory GTPases that arose from a common ancestor early in eukaryotic evolution yet retain extensive structural, biochemical, and functional features. The presence of Arl3 in centrosomes, mitotic spindles, midzones, midbodies, and cilia are all supportive of roles in microtubule-dependent processes. Knockdown of Arl3 by siRNA resulted in changes in cell morphology, increased acetylation of α-tubulin, failure of cytokinesis, and increased number of binucleated cells. We conclude that Arl3 binds microtubules in a regulated manner to alter specific aspects of cytokinesis. In contrast, an excess of Arl2 activity, achieved by expression of the [Q70L]Arl2 mutant, caused the loss of microtubules and cell cycle arrest in M phase. Initial characterization of the underlying defects suggests a defect in the ability to polymerize tubulin in the presence of excess Arl2 activity. We also show that Arl2 is present in centrosomes and propose that its action in regulating tubulin polymerization is mediated at centrosomes. Somewhat paradoxically, no phenotypes were observed Arl2 expression was knocked down or Arl3 activity was increased in HeLa cells. We conclude that Arl2 and Arl3 have related but distinct roles at centrosomes and in regulating microtubule-dependent processes.

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