The kinetics of alkaline phosphatase.

The repressible alkaline phosphatase of Escherichia coli (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) promises to play a role in biochemical genetics comparable to that played by fl-galactosidase (l-5). It is also being used increasingly for class experiments. The evaluation of the important characteristic kinetic constants for this enzyme, however, poses certain technical difficulties (6). The following complications arise. (a) Some buffers, notably Tris, act as phosphate acceptors (7, 8). (b) The enzyme undergoes a marked activation upon incubation with certain buffers (2). (c) The enzymatic activity is sensitive to high ionic strengths. (d) The Michaelis constants (K,) are often so low that when a substrate concentration of this magnitude is investigated, an appreciable fraction is utilized. This makes it hard to determine the true initial velocity. (e) The products of the reaction are potent competitive inhibitors of the substrate. The j&t three sources of error may be readily minimized once they are considered. We wish to point out that the latter two, which reinforce each other, are elegantly handled by the integrated rate equations developed by Niemann and his collaborators (9-l 1). One method they suggested for the presentation of experimental data was to plot [(So) (SJ]/t against l/t.ln [(&)/(SJ] where (So) and (St) are the substrate concentrations initially and at time t, respectively. On such a plot a zero order reaction would yield a horizontal straight line; a first order reaction gives a vertical line cutting the abscissa at a value equivalent to the first order rate constant. Enzymatic reactions in which the products are competitive inhibitors of the substrate yield a series of straight lines with slopes decreasing as the substrate concentration decreases. These lines all appear to emanate from the point V,,,/(l K,/Kp) on the y axis. Km is the substrate Michaelis constant, V,,, is the maximum initial velocity, and K, is the effective product competitive inhibition constant.