Abstract
We have reinvestigated nucleotide binding to actin in order to resolve conflicts regarding the mechanism of nucleotide dissociation and exchange. We present evidence that supports a mechanism for nucleotide binding to actin in which the tightly bound divalent cation (Ca2+ or Mg2+) directly interacts with the bound nucleotide. The dissociation rates of ATP or ADP from actin are limited by the dissociation of the high affinity divalent cation from actin and vary inversely with free Ca2+ or free Mg2+ concentration. The divalent cation concentration range over which attenuation of the ATP dissociation takes place is about 100-fold greater for Mg2+ than that for Ca2+ due to the much slower association rate constant for Mg2+ compared with Ca2+. The relative affinity for ATP versus ADP is 200:1 for Ca-actin in 100 microM free [Ca2+], and 4:1 for Mg-actin in 100 microM free [Mg2+]. Actin without a tightly bound divalent cation has about a 3-fold greater affinity for ATP than ADP. At constant free divalent cation concentration, the rate of nucleotide exchange on actin is described by competitive binding kinetics.
Highlights
We have reinvestigated nucleotide binding to actin ADP binding by actin, which are regulated by the binding of in order toresolve conflicts regarding the mechanism Ca" to an allosteric site of moderate affinity
Reports havebeen published that provide strong evidence that thehigh affinity divalent cation is bound to the@ and y phosphates of the actin-bound ATP
The recentdetermination of the atomic structure of actin including the close relationship between the actin-bound nucleotide and divalent cation forms a structural basis for the present model for nucleotide dissociation from actin
Summary
We present [6] proposed that the Ca-ATPcomplex preferentially associevidencethatsupportsamechanismfornucleotide ates to actin, and that thehigh affinity divalent cation regubinding to actin in which the tightly bound divalent lates nucleotide dissociation, but that thCea2+associated with cation (Ca2o+r M 8 + ) directly interactswith the bound the actin-bound ATP is not the high affinity divalent cation. The relative affinity for ATP versus ADP is 200:1 for Ca-actin in100 p~ free [Ca2+], and4:l for Mg-actin in one (by Frieden and Patane)requires that thefree nucleotide is the species which binds to actin, and the other model (by Nowak et aL) states that the metal-nucleotide complex associates to actinpreferentially. Reports havebeen published that provide strong evidence that thehigh affinity divalent cation is bound to the@ and y phosphates of the actin-bound ATP. Work by Valentin-Ranc andCarlier [13] clearly shows that thebinding
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