Covalent binding of ATP gamma S to the nucleotide-binding site in S14C-actin

We have recently reported on the characterization of beta-actin carrying th e mutation S14C in one of the phosphate-binding loops. The present paper de scribes the attachment of the adenosine 5'-[gamma-thio]-triphosphate (ATP g amma S) to actin containing this mutation. Treatment of S14C-actin with ATP gamma S blocked further nucleotide exchange and raised the thermal stabili ty of the protein, suggesting the formation of a covalent bond between the sulfhydryl on the terminal phosphate of ATP gamma S and cysteine-14 of the mutant actin. The affinity of the derivatized G-actin for DNase I as compar ed to wild-type ATP-actin was lowered to a similar extent as that of ADP.AI F(4)-actin. The derivatized actin polymerized slower than ATP-actin but fas ter than ADP-actin. Under these conditions the bound ATP gamma S was hydrol yzed, suggesting the formation of a state corresponding to the transient AD P.P-i-state. ATP gamma S-actin interacted normally with profilin, whereas t he interaction with actin depolymerizing factor (ADF) was disturbed, as jud ged on the effects of these proteins on actin polymerization. (C) 2000 Fede ration of European Biochemical Societies. Published by Elsevier Science B.V . All rights reserved.