Stereoselectivity of the guanyl-exchangeable nucleotide-binding site of tubulin probed by guanosine 5'-O-(2-thiotriphosphate) diastereoisomers.
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-thiotriphosphate) (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 degrees 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 GTP 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Roychowdhury
S
S
Oklahoma Medical Research Foundation, Oklahoma City 73104.
Gaskin
F
F
D000818:Animals; D001665:Binding Sites; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D013237:Stereoisomerism; D013552:Swine; D013873:Thionucleotides / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D013873:Thionucleotides; D014404:Tubulin; C034883:guanosine 5'-O-(2-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D008274:Magnesium