Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues.
Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.
Carlier
M F
MF
Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France.
Didry
D
D
Simon
C
C
Pantaloni
D
D
D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D005459:Fluorides; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008870:Microtubules / Q000378:metabolism*; D008962:Models, Theoretical; D010710:Phosphates / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D010710:Phosphates; D014404:Tubulin; D006153:Guanosine Diphosphate; C035982:beryllium fluoride; D006160:Guanosine Triphosphate; D001608:Beryllium; D005459:Fluorides