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Covalent binding of ATPγS to the nucleotide‐binding site in S14C‐actin
Author(s) -
Schüler Herwig,
Schutt Clarence E.,
Lindberg Uno,
Karlsson Roger
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01717-8
Subject(s) - actin , actin binding protein , nucleotide , biochemistry , profilin , actina , cysteine , adenosine triphosphate , actin remodeling , chemistry , biology , actin cytoskeleton , cytoskeleton , enzyme , cell , gene
We have recently reported on the characterization of β‐actin carrying the mutation S14C in one of the phosphate‐binding loops. The present paper describes the attachment of the adenosine 5′‐[gamma‐thio]‐triphosphate (ATPγS) to actin containing this mutation. Treatment of S14C‐actin with ATPγS blocked further nucleotide exchange and raised the thermal stability of the protein, suggesting the formation of a covalent bond between the sulfhydryl on the terminal phosphate of ATPγS and cysteine‐14 of the mutant actin. The affinity of the derivatized G‐actin for DNase I as compared to wild‐type ATP‐actin was lowered to a similar extent as that of ADP.AlF 4 ‐actin. The derivatized actin polymerized slower than ATP‐actin but faster than ADP‐actin. Under these conditions the bound ATPγS was hydrolyzed, suggesting the formation of a state corresponding to the transient ADP.P i ‐state. ATPγS‐actin interacted normally with profilin, whereas the interaction with actin depolymerizing factor (ADF) was disturbed, as judged on the effects of these proteins on actin polymerization.