A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (P-i), in particular to follow the release of P-i in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli phosphate binding protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled protein and unattached MDCC. The labeled protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of P-i, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda(max)/lambda(max)) when complexed with P-i at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. P-i binds tightly (K-d similar to 0.1 mu M) and rapidly (1.36 x 10(8) M(-1) s(-1)) and the dissociation rate constant is 21 s(-1), at pH 7.0, low ionic strength, 22 degrees C. A variety of phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 mu M or higher concentration. ATP weakly inhibited the P-i-induced fluorescence change, indicating that it binds at least 3000-fold weaker than P-i, Because P-i is a widespread contaminant, the probe is used in conjunction with a ''P-i mop'', consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free P-i from solutions by its conversion to ribose 1-phosphate. Because the equilibrium constant of this reaction is >100, free P-i can be reduced below 0.1 mu M The probe was used to measure the rate of P-i release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent P-i release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid P-i release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a K-m with respect to actin of 26 mu M. The data at 22 degrees C and at [actin] that is close to saturating (45-50 mu M) fitted a kinetic scheme, comparable with that obtained from other workers, with the following rate constants: ATP binding, k(+1) = 6 x 10(6) M(-1) s(-1), k(-1) = 7 s(-1); ATP cleavage, k(+2) = 30 s(-1), k(-2) = 15 s(-1); P-i release, k(+3) = 45 s(-1), k(-3) = 0 s(-1); ADP release, fast.
