Phosphorus-31 nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH

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Abstract
31P NMR was used to directly observe the binding of Pi. to alkaline phosphatase (EC-3.1.3.1) [Escherichia coli]. Evidence for the tight binding of 1.5-2.0 mol of Pi/dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, 1 predominating above pH 7 and representing the non-covalent E.cntdot.Pi complex and the other predominating below pH 5 and representing the covalent E-Pi complex. The 31P NMR line width of the E.cntdot.Pi complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.