A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps

The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.