Applied Biochemistry and Biotechnology

, Volume 171, Issue 6, pp 1454–1464 | Cite as

Tailoring the pH Dependence of Human Non-pancreatic Secretory Phospholipase A2 by Engineering Surface Charges

Article
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Abstract

Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) catalyzes the sn-2 acyl hydrolysis of phospholipids. It was reported that hnpsPLA2 is involved in various diseases like inflammation, cancer, and so on. This enzyme also exhibits anti-bacterial and anti-virus activities. It is active over a broad pH range, with higher activity at alkaline conditions. In order to make it suitable as a potential bactericide, high activity at neutral pH is preferable. We have tried to tailor the pH dependence of hnpsPLA2 activity by replacing its surface charged residues. Three surface charge replacements, Arg42Glu, Arg100Glu, and Glu89Lys, showed increased activities at neutral pH, which are 2.3, 2.8, and 2.3 times that of the wild-type enzyme at pH 7. Both the positive-to-negative and negative-to-positive mutations lowered the optimum enzymatic reaction pH of hnpsPLA2, indicating that the enzyme pH profile depends on a delicate balance of charged residues. The activity changes are in good agreement with the recently proposed calcium-coordinated catalytic triad mechanism. This study also provides a general means of enhancing hnpsPLA2 activity at low pH.

Keywords

Phospholipase A2 Protein engineering Enzyme reaction optimum pH Catalytic mechanism The catalytic triad mechanism The calcium-coordinated oxyanion mechanism 

Abbreviations

hnpsPLA2

Human non-pancreatic secretory phospholipase A2

IBS

Interface binding site

CD

Circular dichroism

ANS

1-anilinonaphthalene-8-sulfonate

DMPC

1,2-dimyristoyl-sn-glycero-3-phosphocholine

GIIAPLA2

Group IIA phospholipase A2

Vmax

The maximum velocity of the enzymatic activity

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Notes

Acknowledgments

This work was supported in part by the National Natural Science Foundation of China and the Ministry of Science and Technology of China.

Supplementary material

12010_2013_437_MOESM1_ESM.pdf (246 kb)
ESM 1 (PDF 246 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular EngineeringPeking UniversityBeijingChina
  2. 2.Center for Quantitative BiologyPeking UniversityBeijingChina

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