Stereoselectivity of the guanyl-exchangeable nucleotide-binding site of tubulin probed by guanosine 5'-O-(2-thiotriphosphate) diastereoisomers

Publisher Website
Google Scholar
Abstract
The active site of the exchangeable nucleotide-binding site of tubulin was studied by using diastereoisomers A (SP) and B (RP) of guanosine 5''-O-(2-thiophosphate) (GTP.beta.S) where the phosphorus atom to which sulfur is attached is chiral. Turbidimetric measurements were used to follow kinetics, and electron microscopy was used to evaluate polymeric forms. Both isomers at 0.5 mM promoted the assembly of tubulin in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid, 30%glycerol, 3 mM MgCl2, and 1 mM EGTA, pH 6.6, 23-37.degree. C. GTP.beta.S(A) promoted assembly into microtubules, although a few bundles were also found by electron microscopy. However GTP.beta.S(B) induced assembly of tubulin into bundles of sheets and microtubules. As expected, 0.5 mM GTP induced tubulin to assemble into microtubules, thin sheets, and a few bundles. Both GTP and GTPY.beta.S(A) were hydrolyzed in the tubulin polymers. However, more than 95% of the bound GTP.beta.S(B) was not hydrolyzed. Higher concentrations of GTP.beta.S(B), i.e.,1 mM, also induced bundles of sheets and microtubules, with 86% of the thionucleotide bound as the triphosphate. The GTP.beta.S(B)-induced polymers were considerably more cold stable than the GTB.beta.S(A)-induced microtubules, which were more cold stable than GTP-induced polymers. Mg(II) (2-5 mM)had minimal effects on the structures induced by GTP.beta.S(A) or -(B) isomers in the tubulin assembly system. However, at 1 mM Mg(II), no assembly was found with GTP.beta.S(A) and tubulin. The assembly studies on GTP.beta.S(A) and -(B) with microtubule protein (tubulin plus microtubule-associated proteins) showed similar kinetics at 0.5 mM Mg(II) in buffer without glycerol, and microtubules were the major structures. Most of the bound nucleotide was hydrolyzed. With varyingMg(II) and GTP or GTP.beta.S(B) concentrations, only microtubules were found. However, different Mg(II)/GTP.beta.S(A) ratios resulted in different polymeric forms with rings and cross-linked rings being the predominant polymers ast high Mg(II) or GTP.beta.(A) concentrations. These studies demonstrate stereoselectivity at the .BETA. phosphorus of the exchangeable nulcleotide-binding site on tubulin. The GTP.beta.S(A) isomer is a better GTP analogue than GTP.beta.S(B) in the tubulin assembly system, whereas GTP.beta.S(B) is a better GTP analogue in the microtubule protein assembly system.