Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2,4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate 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 Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were 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 were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; 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 solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values 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 reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (ϕ) for deuterium for the transition state (ϕT) increased when the pH of the solution was raised. At pL 11.0, the ϕT was 1.07 in reverse micelles, which corresponds to the inverse-isotope effect of the reaction in this solvent system. Normal viscosity effects on kcat and kcat/Km were observed in aqueous solution, corresponding to a diffusional controlled physical step as the rate-limiting step. We propose that the rate-limiting step of the hydrolytic reaction changes from phosphate releasing in aqueous solution to a covalent phosphorylation or dephosphorylation step in reverse micelles.
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February 1998
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Research Article|
February 15 1998
Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles
Ter-Mei HUANG;
Ter-Mei HUANG
1Graduate Institutes of Biochemistry and Life Sciences, National Defense Medical Centre, Taipei, Taiwan, Republic of China
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Hui-Chih HUNG;
Hui-Chih HUNG
1Graduate Institutes of Biochemistry and Life Sciences, National Defense Medical Centre, Taipei, Taiwan, Republic of China
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Tsu-Chung CHANG;
Tsu-Chung CHANG
1Graduate Institutes of Biochemistry and Life Sciences, National Defense Medical Centre, Taipei, Taiwan, Republic of China
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Gu-Gang CHANG
Gu-Gang CHANG
1
1Graduate Institutes of Biochemistry and Life Sciences, National Defense Medical Centre, Taipei, Taiwan, Republic of China
1To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
July 04 1997
Revision Received:
September 22 1997
Accepted:
October 07 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 330 (1): 267–275.
Article history
Received:
July 04 1997
Revision Received:
September 22 1997
Accepted:
October 07 1997
Citation
Ter-Mei HUANG, Hui-Chih HUNG, Tsu-Chung CHANG, Gu-Gang CHANG; Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles. Biochem J 15 February 1998; 330 (1): 267–275. doi: https://doi.org/10.1042/bj3300267
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