Possible kinetic mechanism of human placental alkaline phosphatase in vivo as implemented in reverse micelles

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Abstract
Human placental alkaline phosphatase is an integral membrane protein. It catalyzes the hydrolysis of phosphoester linkage of a broad-range substrate. We have embedded the enzyme in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. Linear Lineweaver-Burk and Eadie-Hofstee plots for the substrate and linear Arrhenius plot for the temperature-dependent enzyme reaction were obtained in reverse micelles suggesting that the substrate diffusion limitation was not a rate-limiting step in the system and exchange of materials between reverse micelles was very rapid. The catalytic constant (kcat) of the enzyme was decreased, and the Km for the substrate was increased in reverse micelles, both in an exponential way with the [H2O]/ AOT] ratio (omega O). The enzyme was more stable in reverse micelles than in aqueous solution at 30 degrees C but was unstable at higher temperature (65 degrees C). The activation energy of the enzyme in reverse micelles was 46.5 +/- 2.6 kJ/mol, which was about 20 kJ/mol higher than that in aqueous solution and reflected in the lower Kcat value at low omega O. The binding affinity between the substrate 4-nitrophenyl phosphate and the enzyme in reverse micelles was decreased as implemented by the higher Km and higher Ki for phosphate values. In aqueous solution, the log kcat/pH plot suggested that amino acid residues with pKa values of 9.03 +/- 0.03 and 11.37 +/- 0.10 are involved in catalysis. The former should be deprotonated and the latter should be protonated for the reaction to proceed. In reverse micelles, both the above-mentioned pKa values were detected. However, both groups have to deprotonated to give the optimum catalytic function. In aqueous solution, the enzyme's specificity was highly dependent on pH and buffer. The pKa value of the amino acid residues involved in substrate binding was found to be 8.69 +/- 0.07 in carbonate buffer, but changed to 9.80 +/- 0.06 in Tris buffer. Our results suggested that the rate-limiting step of the enzyme-catalyzed reaction may be changed from phosphate releasing in aqueous solution to another critical step in the reverse micelles.