Studies on the nocodazole-induced GTPase activity of tubulin.
The tubulin dimer contains two guanine nucleotide binding sites, a nonexchangeable site occupied by GTP and an exchangeable site (E-site) occupied by GTP or GDP. Under the conditions used in this study the E-site GTP was hydrolyzed at a rate of 8 x 10(-5) s(-1) at 37 degrees C. This rate is stimulated four- to fivefold by nocodazole, an antimitotic drug. We studied the characteristics of this drug-stimulated reaction to learn more about the hydrolytic center of tubulin. The reaction, studied using single turnover kinetics, i.e., in the absence of added GTP, has a pH optimum of 6.6 to 7.2 and an activation energy of 88 kJ x mol(-1). It is first-order with respect to tubulin-GTP, indicating that hydrolysis is not dependent on an aggregation process. Divalent cations stimulate the hydrolysis three- to six-fold over the rate in the presence of EDTA. The reaction has a requirement for Na+ that is not satisfied effectively by other monovalent cations. In contrast, Na+ and K+ are almost equally effective in the tubulin assembly reaction. Different purine nucleoside triphosphates can bind to the E-site and are hydrolyzed. GTP and ITP are hydrolyzed at equivalent rates and XTP and ATP are hydrolyzed at a rate about half as fast. Hydrolysis is inhibited by the reagents diethylpyrocarbonate and N-ethylmaleimide, although the GTPase activity is less sensitive than the assembly reaction. The reaction rate in D2O is twice the rate in H2O. This inverse isotope effect suggests the involvement of a sulfhydryl group in the rate-limiting step in the reaction.
Mejillano
M R
MR
Department of Biochemistry, University of Kansas, Lawrence 66045-2106, USA.
Shivanna
B D
BD
Himes
R H
RH
D000818:Animals; D002021:Buffers; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002417:Cattle; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008870:Microtubules / Q000201:enzymology; D015739:Nocodazole / Q000494:pharmacology; D012997:Solvents; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
D002021:Buffers; D002413:Cations, Divalent; D002414:Cations, Monovalent; D012997:Solvents; D013439:Sulfhydryl Reagents; D014404:Tubulin; D006639:Histidine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D015739:Nocodazole