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The correlation between the time courses of pure tubulin assembly and accompanying guanosine 5'-triphosphate (GTP) hydrolysis has been studied at different tubulin concentrations in the range where the rate of assembly varies with a strong cooperativity. One GTP molecule was found hydrolyzed per molecule of tubulin dimer incorporated in the microtubule. This hydrolysis was not strictly coupled to polymerization and occurred in a subsequent step. Consequently, in the first stages of assembly, tubulin-GtP complex is the transient major constituent of microtubules. Kinetic data of GTP hydrolysis have been treated within a model of two consecutive first-order reactions: tubulin-GTPfree k1 leads to tubulin-GTPMT k2 leads to tubulin-GDPMT + Pi GTP hydrolysis proceeded at an intrinsic rate k2 = 0.25 min-1 independent of tubulin concentration. Simultaneous measurements of polymerization, GTPase activity, and incorporation of 3HGTP followed by unlabeled GTP chase indicated that before its hydrolysis GTP bound to microtubules was exchangeable while after hydrolysis GDP remained locked in the E site. The possibility is discussed that after assembly tubulin undergoes a conformation change which could trigger GTP hydrolysis and sequestration of GDP.
Carlier et al. (Tue,) studied this question.