The reaction of alkaline phosphatase (APase) with the complexes of myo-inositol hexakisphosphate (IHP) and various cations at pH 7.2 results in a decrease in activity. Singly, neither IHP nor metal ions induce such changes. IHP-Mn(II) complexes were the least effective. Using the ions of nickel or cadmium, activity was reduced by > 95%. A similar large decrease (> 99%) was seen previously in the reaction of APase with IHP-Cu(II) complexes. With Co(II) and IHP as reactants, the activity was reduced to 10–12% of that of the native enzyme. When the apoprotein, prepared by reaction of the enzyme with either EDTA or 1,10-phenanthroline, was titrated with Co(II), the activity was equal to that resulting from the reaction of the enzyme with IHP-Co(II) complexes. Titration with zinc restored 95% of the original activity. The products are metal-substituted derivatives in which the resident catalytic (A-site) zinc ions, at least, are replaced by the cation of the IHP complex that was used. The rates of such reactions were fastest with the complexes of Cu(II) and Cd(II) (0.12 min −1), less so with Co(II) as the ion (0.056 min −1), and slowest with complexes of nickel and manganese (0.01 min −1). In every case, the rate of reaction, but not its extent of change, was inhibited by zinc ions that reduced rate constants to 0.0014–0.0054 min −1. Magnesium ions had no effect.Likewise, Mn(II), with but one exception, did not affect the reactions. When present along with IHP-Ni(II) complexes, the rate was increased and the enzyme activity further decreased. If Zn(II) was also present, this enhancement was eliminated. All changes in enzyme activity were reversible by treatment with EDTA followed by reconstitution with zinc. Approximately 95% conversion to the original activity could be attained. Reactivation of modified APase preparation also could be attained, in some cases, by pre-incubation with Zn(II) at pH 8. For example, conversion of the Cd(II)-substituted APase to the zinc enzyme was rapid and complete in 15 min. With the Cu(II)-substituted derivative, reactivation was much slower. Incubation with zinc ions had little or no effect on other Me(II)-substituted APase preparations. Co-APase and Cu-APase, prepared from the apoprotein, behaved similarly to their respective “counterpart product” of the approriate metal ion-exchange reaction. In contrast, Co-APase, but not Cu-APase, could be converted to the zinc enzyme by incubation with IHP-Zn(II) complexes at pH 7.2. The reaction rate of the various metal-substituted APase preparations with EDTA varied with the IHP-Me(II) used in its formation. The observed rate constants for Zn-APase and Co-APase were faster than those in the presence of their respective IHP-cation complex. IHP-Me(II) complexes are thuscompetitive inhibitors due to their binding at or near the active sites. In support of previous conclusions, only a small fraction of the total complexes present in the reaction solution appears to participate in the reaction. It is suggested that only a chelate complex designated as (IHP-Me) ∗ is the actual reactant that forms a transient ternary complex with the A-site zinc atom and the apoprotein. Its concentration determines the reaction rate leading to a Me(II)-substituted derivative. In this reaction, (IHP-Me) ∗ is proposed as the acceptor for the catakytic zinc ion through its provision of a binding site whose affinity for zinc is greater than that of the enzyme in the transient activated state. In the reaction of APase with IHP-Co(II) complexes, saturation kinetics similar to a Michaelis-Menten-type reaction was obtained. With IHP and Co(II) at a 1:1 ratio, the concentration of (IHP-Co) ∗ was determined to be only 0.94% of the total complexes present assuming that 1 mol-mol −1 of active site is required. At the higher ratio of Co(II):IHP of 3:1, the (IHP-Co) ∗ concentration declines to 0.54%. It approached zero as the ratio was further increased or the ratio became < 1. Assuming that one (IHP-Co) ∗ molecule reacts with ony one catalytic site in the formation of the intermediate state, the association constant has been kinetically determined to be 16.8 × 10 6 M −1. For the reaction of APase with IHP and Cu(II), the concentration of the (IHP-Cu) ∗ species was also small (0.18%) with K assoc = 6.7 × 10 5 M −1. Aspects of the possible nature of the (IHP-Me) ∗ structure have been discussed. For reasons that give advantage to its proposed functional role in the mechanism of the reaction, a complex with two bound cations, (IHP-Me 2) ∗, is suggested.
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Dec 27, 2015
Qing-Yun Chen + 5
Open Access