ACCURATE KINETIC MODELING OF ALKALINE-PHOSPHATASE IN THE ESCHERICHIA-COLI PERIPLASM - IMPLICATIONS FOR ENZYME PROPERTIES AND SUBSTRATE DIFFUSION

Alkaline phosphatase in the periplasm of Escherichia coli presents man y of the complex factors that may influence enzymes in vivo. These inc lude an environment that contains a high enzyme concentration, is dens ely populated with other macromolecules, and is separated from other c ompartments by a partial diffusion barrier. A previous study provided a partial description of this situation and developed a model that uti lized kinetic behavior to estimate the permeability of the outer membr ane [Martinet, M. B., et al., (1992) Biochemistry 31, 11500]. This stu dy extends that description to provide a complete model for the enzyme at all substrate levels. Some of the parameters needed for complete m odeling include the following: outer membrane permeability to the subs trate and product, catalytic efficiency of the enzyme, number of enzym es per cell, and effects of the reaction product (an inhibitor) on the enzyme. The theoretical model fit the data quite well over a wide ran ge of values for each of these parameters. The best fit of theory with experimental data required that the rate constant for product escape from the periplasm was 4-fold greater than that for substrate entry. T his correlated with the relative sizes of the substrate and product. T he excellent fit of theory and results suggested that alkaline phospha tase and its substrate were unaffected by the solution conditions in t he periplasm. That is, the catalytic parameters (k(cat) and K-M), dete rmined for the enzyme in dilute solution, appeared to be unchanged by the conditions in the periplasm. The major factor that altered the kin etic behavior was the combined effect of the permeability barrier and the dense population of enzyme molecules in the periplasm. Given the l arge impact of these parameters on reaction properties, the excellent fit of theory and results was striking. Overall, this study demonstrat ed that enzyme action in the complex biological environment can be acc urately modeled, if all factors that influence enzyme behavior are kno wn.