Abstract
Oxovanadium(IV) ion (VO 2+) and vanadium(V) ion (VO 3 −) are potent competitive inhibitors of the Escherichia coli alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate. The dissociation constants ( K i ) of the enzyme-inhibitor complexes have been determined as a function of pH over the pH range 7.0–10.0. Vanadium(V) ( K i = 2.2 × 10 −6 m, pH 8.0, Tris buffer) binds about as well as inorganic phosphate and has a pH- K i profile similar to that of phosphate. Oxovanadium(IV) ion binds about one to two orders of magnitude better than phosphate over this pH range ( K i = 4 × 10 −7 m, pH 8.0, barbital buffer). Theorell-Yonetani plots show that vanadium(V) binds mutually exclusively with inorganic phosphate, and VO 2+ also appears to bind in this manner at lower inhibitor concentrations. Tris buffer lowers the effectiveness of VO 2+ as an inhibitor by a factor of four to five. These observations indicate that VO 3 − can bind quite specifically at the phosphate-binding site on the enzyme. The more potent inhibition by VO 2+ suggests that the enzyme-complexed inhibitor may have some resemblance to the metastable intermediate formed during the hydrolysis of phosphate esters.
Published Version
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