Kinetic analysis of guanosine 5'-triphosphate hydrolysis associated with tubulin polymerization.
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-GTP]free k1 leads to [tubulin-GTP]MT k2 leads to [tubulin-GDP]MT + [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 [3H]GTP 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.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008870:Microtubules / Q000648:ultrastructure; D010761:Phosphorus Radioisotopes; D013552:Swine; D014316:Tritium; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D014404:Tubulin; D014316:Tritium; D006160:Guanosine Triphosphate