Nucleotide binding to actin. Cation dependence of nucleotide dissociation and exchange rates

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.