The Nature of Negative Cooperativity in Alkaline Phosphatase

The nature of strong negative cooperativity displayed by alkaline phosphatase from Escherichia coli was investigated by alternative substrate and product inhibition studies, and by catalytic rate constant (kcat) measurements. To see whether the idled subunit in this dimeric enzyme plays a mechanistic role in the overall catalysis, the flip-flop model proposed by Lazdunski et al. ((1971) Eur. J. Biochem 20, 124) was examined. In this mechanism, the dephosphorylation of the phosphoryl enzyme intermediate at one subunit is supposedly facilitated by the binding of substrate to the second subunit. Kinetic theory predicts that for such a model, when an alternative substrate is present at fixed levels, double reciprocal plots with respect to the substrate should be nonlinear. Our kinetic data, however, yielded linear competitive inhibition patterns. When the alternative substrate is present at constant ratios to the substrate, linear intersecting double reciprocal plots are predicted. Instead, parallel plots were obtained for three substrate-alternative substrate pairs. Nonlinear, noncompetitive inhibition by the product inorganic phosphate is predicted for the flip-flop model, but linear competitive inhibition was observed. In addition, the kcat determined at pH 8,25”C in 0.1 M Tris-HC1 is 27 s”, which agrees very well with the “off-rate” of inorganic phosphate, -25 s-l, determined by Hull et aL ((1976) Biochemistry 15,1547) using the NMR technique under identical conditions. All the kinetic experiments are consistent with a mechanism in which the idled subunit plays no discernible role. They also indicate that the flip-flop mechanism is not operative in the alkaline phosphatase system. The dependence of kcat of alkaline phosphatase on the concentration of ”is-HC1 at 10°C and 25”C, pH 8, showed saturation phenomena. Analysis of the data revealed that an enzyme-Tris-HC1 complex was involved, and that the observed kcat could be resolved into two components corresponding to the product release rate constants for inorganic phosphate and Trisphosphate.