INTERACTION OF TUBULIN WITH GUANOSINE 5'-O-(1-THIOTRIPHOSPHATE) DIASTEREOISOMERS - SPECIFICITY OF THE ALPHA-PHOSPHATE BINDING REGION

The exchangeable nucleotide-binding site of tubulin has been studied u sing diastereoisomers A (S-p) and B (R(p)) of guanosine 5'-O-(1-thiotr iphosphate) (GTP alpha S) in which the phosphorus atom to which sulfur is attached is chiral. GTP alpha S(A) (10 mu M) nucleated assembly of purified tubulin (20 mu M) into microtubules in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid with 3 mM Mg2+ and 1 mM EGTA, p H 6.6 at 37 degrees C. With 0.2 mM GTP alpha S(A), the critical concen tration (Cc; minimum protein concentration required for assembly) was 8 mu M tubulin. Neither 0.2 mM GTP nor GTP alpha S(B) promoted microtu bule assembly in buffer with 0.5-6.75 mM Mg2+ and 20-70 mu M tubulin. The Cc values for GTP alpha S(A)-induced assembly of tubulin in buffer with 30% glycerol and of microtubule protein (tubulin and microtubule -associated proteins) in buffer were lower than for GTP. GTP alpha S(A )-induced microtubules were more stable to the cold and to Ca2+. GTP a lpha S(A) and GTP but not GTP alpha S(B) bound tightly to tubulin at 4 degrees C. Although GTP alpha S(B) did not nucleate assembly, it did bind to tubulin since it was incorporated into the growing microtubule . Both isomers were hydrolyzed in the microtubules. These studies show that GTP alpha S(A) promotes tubulin assembly better than GTP and GTP alpha S(B) and that there is stereoselectivity at the cu-phosphate bi nding region of tubulin. The stereoselectivity may be due to different MgGTP alpha S(A) and -(B) interactions with tubulin.