Effect of Methanol on the Activity and Conformation of Acid Phosphatase from the Prawn Penaeus penicillatus

Q.-X. Chen^1*, R.-Q. Zhang^2,3, X.-Z. Xue¹, P.-Z. Yang¹, S.-L. Chen¹, and H.-M. Zhou^2,3*

¹Department of Biology, Xiamen University, Xiamen 361005, China

²Department of Biological Science and Biotechnology, Tsinghua University, China; fax: +8610 62785505; E-mail: zhm-dbs@mail.tsinghua.edu.cn

³Center for Ocean Science and Engineering, Tsinghua University, Beijing 100084, China

^* To whom correspondence should be addressed.

Received May 11, 1999

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Prawn (Penaeus penicillatus) acid phosphatase (EC 3.1.3.2) catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in sea water on the enzyme activity results in the loss of the biological function of the enzyme, which leads to disruption of phosphate metabolism in cells. This paper analyzes the effects of methanol on the activity and conformation of prawn acid phosphatase. The results show that low concentrations of methanol can lead to reversible inactivation. Inhibition of the enzyme by methanol is classified as non-competitive inhibition, and the inhibition constant (K_i) is 8.5%. Conformational changes of the enzyme molecule in methanol solutions of different concentrations were measured using fluorescence emission, differential UV-absorption, and circular dichroism spectra. Increased methanol concentrations caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with methanol had two negative peaks, at 222 and 270 nm, and a positive peak at 236 nm. The changes in the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed some. These results suggest that methanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.

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KEY WORDS: prawn (Penaeus penicillatus), acid phosphatase, catalytic activity, conformation, methanol

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