The Phosphate Content of Escherichia coli Alkaline Phosphatase and Its Effect on Stopped Flow Kinetic Studies

Alkaline phosphatase purified from Escherichia coli contains up to 2 molecules of tightly bound inorganic phosphate per enzyme molecule. Removal of this endogenous ligand alters the stopped flow traces obtained for the hydrolysis of substrates at pH 5.5 and at pH 8.0. The pre-steady state burst transient of product release normally seen at pH 5.5 (τ-1 = 20 s-1) is transformed into an instantaneous burst of equal amplitude; an equivalent instant phase is created at pH 8.0, where no burst normally is seen. Addition of 1.5 eq of phosphate to phosphate-free enzyme restores the stopped flow trace completely to the normal burst transient. The maximum burst amplitude seen at all stoichiometries of enzyme preincubated with phosphate at pH 7.4 before reaction with substrate at pH 5.5 is 1.4 to 1.6 moles of product released per mole of enzyme and not 1.0 as previously reported. This amplitude is decreased if enzyme is preincubated with endogenous or added phosphate at pH values below 6.5. These results suggest that: (a) during the steady state turnover of substrate, the enzyme shuttles between two conformations, one of which binds substrate preferentially and catalyzes substrate hydrolysis and one of which binds phosphate preferentially and has no catalytic activity; the former conformation is thermodynamically preferred in the absence of phosphate; and (b) the active sites on the alkaline phosphatase molecule may be equivalent and independent, at least in the pre-steady state.