SOLVENT KINETIC ISOTOPE EFFECTS OF HUMAN PLACENTAL ALKALINE-PHOSPHATASE IN REVERSE MICELLES

Human placental alkaline phosphatase was embedded in a reverse micella r system prepared by dissolving the surfactant sodium bis(2-ethylhexyl ) sulphosuccinate (Aerosol-OT) in 2,2,4-trimethylpentane. This microem ulsion system provides a convenient instrumental tool to study the pos sible kinetic properties of the membranous enzyme in an immobilized fo rm. The pL (pH/p(2)H) dependence of hydrolysis of 4-nitrophenyl phosph ate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were H-a-bell sh aped in the acidic region but reached a plateau in the basic region in which two pK(a) values of 9.01-9.71 and 9.86-10.48, respectively, wer e observed in reverse micelles. However, only one pKa value of 9.78-10 .27 in aqueous solution was detected. Profiles of log V/K versus pL we re bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pK(a) 9.10-9.44 in aqueous sol ution and 8.07-8.78 in reverse micelles must be dehydronated for the r eaction to reach an optimum. The V/K value shifted to a lower value up on dehydronation of a pK(a) value of 9.80-10.62 in aqueous solution an d 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects wer e measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effe ct; at pL 10.4, the log V/K value was identical in water and deuterium . The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous s olution and 1.16 in reverse micelles. At pL 11.0 at which the log V va lues reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in rever se micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the cat alytic reaction. The isotopic fractionation factor (phi) for deuterium for the transition state (phi(T)) increased when the pH of the soluti on was raised. At pL 11.0, the phi(T) was 1.07 in reverse micelles, wh ich corresponds to the inverse-isotope effect of the reaction in this solvent system. Normal viscosity effects on k(cat) and k(cat)/K-m were observed in aqueous solution, corresponding to a diffusional controll ed physical step as the rate-limiting step. We propose that the rate-l imiting step of the hydrolytic reaction changes from phosphate releasi ng in aqueous solution to a covalent phosphorylation or dephosphorylat ion step in reverse micelles.