Kinetics of nucleotide and metal ion interaction with G-actin

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Abstract
The kinetics of interaction of Ca2+ ions and nucleotides with G-actin have been investigated by making use of the enhancement of 1,N6-ethenoadenosine 5''-triphosphate (.epsilon.ATP) fluorescence on binding to actin, the enhancement of 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline (Quin-2) fluorescence on binding to Ca2+, and the sensitivity of the fluorescence of an N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine (1,5-AEDANS) group on Cys-374 to metal ion binding. It is concluded that metal ion dissociation is the rate-limiting step in nucleotide dissociation (0.016 s-1 for Ca2+ at pH 7.2 and 21.degree. C) and that earlier conclusions that metal ion release is relatively fast and subsequent nucleotide release slow are incorrect. Results presented here and obtained by others on the metal ion concentration dependence of the effective rate of nucleotide exchange can be interpreted in the light of this conclusion in terms of a limiting rate which corresponds to that of metal ion release and an "apparent" dissociation constant for Ca2+ which is without direct physical significance. This apparent dissociation constant is more than 2 orders of magnitude greater than the real dissociation constant of Ca2+ from the Ca.sbd.actin.sbd.ATP complex, which was estimated to be 2 .times. 10-9 M from a titration with Quin-2. Confirmation that the rate of Ca2+ release is rate limiting both in nucleotide dissociation reactions and in replacement of Ca2+ by Mg2+ was obtained with 1,5-AEDANS.sbd.actin, since both the replacement of Ca2+ by Mg2+ and the removal of Ca2+ to give the actin.sbd.ATP complex occurred at the same (slow) rate. Metal ion exchange kinetics (Mg2+/Ca2+) can be explained by use of the simplest possible kinetic model of interaction of both metal ions and with appropriate rate constants for the forward and reverse rate constants of metal ion binding. In particular, the rate of dissociation of Mg2+ is about 1 order of magnitude lower than that for Ca2+. The data obtained, together with thermodynamic considerations, lead to the conclusion that the tightly bound Ca2+ cannot be bound as a complex with ATP. Kinetic evidence suggesting that a meal ion-nucleotide complex is recognized in the binding reaction at relatively high metal ion concentration thus requires that this is a further metal ion, possibly one of the "weakly" bound ones.