Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Mar 15;346(Pt 3):679–690.

Mapping the catalytic pocket of phospholipases A2 and C using a novel set of phosphatidylcholines.

J J Caramelo 1, J Florín-Christensen 1, M Florín-Christensen 1, J M Delfino 1
PMCID: PMC1220900  PMID: 10698694

Abstract

A set of radioiodinatable phosphatidylcholines (PCs) derivatized with the Bolton-Hunter reagent (BHPCs) was synthesized to probe the substrate recognition and activity of phospholipases. A common feature of this series is the presence of a bulky 4-hydroxyphenyl group at the end of the fatty acyl chain attached to position sn-2. The distance between the end group and the glycerol backbone was varied by changing the length of the intervening fatty acyl chain (3-25 atoms). Except for the shortest, this chain includes at least one amide linkage. The usefulness of this series of substrates as a molecular ruler was tested by measuring the hydrolytic activities of Naja naja naja phospholipase A(2) (PLA(2)) and Bacillus cereus phospholipase C (PLC) in Triton X-100 micelles. The activity of PLA(2) proved to be highly dependent on the length of the fatty acyl chain linker, the shorter compounds (3-10 atoms) being very poor substrates. In contrast, the PLC activity profile exhibited much less discrimination. In both cases, PCs with 16-21 atom chains at position sn-2 yielded optimal activity. We interpret these findings in terms of fatty acyl chain length-related steric hindrance caused by the terminal aromatic group, affecting the activity of PLA(2) and, to a smaller extent, that of PLC. This notion agrees with the more extended recognition of aliphatic chains inside the narrow channel leading to the catalytic site in the former case. Molecular models of these substrates bound to PLA(2) were built on the basis of the crystallographic structure of Naja naja atra PLA(2) complexed with a phospholipid analogue. Docking of these substrates necessarily requires the intrusion of the bulky 4-hydroxyphenyl group inside the binding pocket and also the failure of the amide group to form hydrogen bonds inside the hydrophobic substrate channel.

Full Text

The Full Text of this article is available as a PDF (299.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Burley S. K., Petsko G. A. Amino-aromatic interactions in proteins. FEBS Lett. 1986 Jul 28;203(2):139–143. doi: 10.1016/0014-5793(86)80730-x. [DOI] [PubMed] [Google Scholar]
  4. Burley S. K., Petsko G. A. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science. 1985 Jul 5;229(4708):23–28. doi: 10.1126/science.3892686. [DOI] [PubMed] [Google Scholar]
  5. DeBose C. D., Burns R. A., Jr, Donovan J. M., Roberts M. F. Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity? Biochemistry. 1985 Mar 12;24(6):1298–1306. doi: 10.1021/bi00327a005. [DOI] [PubMed] [Google Scholar]
  6. Deems R. A., Eaton B. R., Dennis E. A. Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes. J Biol Chem. 1975 Dec 10;250(23):9013–9020. [PubMed] [Google Scholar]
  7. Delfino J. M., Florín-Christensen J., Florín-Christensen M., Richards F. M. Differential hydrolysis of immobilized phosphatidylcholines by phospholipases A2 and C. Biochem Biophys Res Commun. 1994 Nov 30;205(1):113–119. doi: 10.1006/bbrc.1994.2637. [DOI] [PubMed] [Google Scholar]
  8. Dennis E. A. Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions. Arch Biochem Biophys. 1973 Oct;158(2):485–493. doi: 10.1016/0003-9861(73)90540-7. [DOI] [PubMed] [Google Scholar]
  9. Dennis E. A., Rhee S. G., Billah M. M., Hannun Y. A. Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J. 1991 Apr;5(7):2068–2077. doi: 10.1096/fasebj.5.7.1901288. [DOI] [PubMed] [Google Scholar]
  10. Exton J. H. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta. 1994 Apr 14;1212(1):26–42. doi: 10.1016/0005-2760(94)90186-4. [DOI] [PubMed] [Google Scholar]
  11. Gijón M. A., Leslie C. C. Regulation of arachidonic acid release and cytosolic phospholipase A2 activation. J Leukoc Biol. 1999 Mar;65(3):330–336. doi: 10.1002/jlb.65.3.330. [DOI] [PubMed] [Google Scholar]
  12. Gupta C. M., Radhakrishnan R., Khorana H. G. Glycerophospholipid synthesis: improved general method and new analogs containing photoactivable groups. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4315–4319. doi: 10.1073/pnas.74.10.4315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hansen S., Hough E., Svensson L. A., Wong Y. L., Martin S. F. Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog. J Mol Biol. 1993 Nov 5;234(1):179–187. doi: 10.1006/jmbi.1993.1572. [DOI] [PubMed] [Google Scholar]
  14. Hansen S., Hough E., Svensson L. A., Wong Y. L., Martin S. F. Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog. J Mol Biol. 1993 Nov 5;234(1):179–187. doi: 10.1006/jmbi.1993.1572. [DOI] [PubMed] [Google Scholar]
  15. Hough E., Hansen L. K., Birknes B., Jynge K., Hansen S., Hordvik A., Little C., Dodson E., Derewenda Z. High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus. Nature. 1989 Mar 23;338(6213):357–360. doi: 10.1038/338357a0. [DOI] [PubMed] [Google Scholar]
  16. Hubbell W. L., McConnell H. M. Molecular motion in spin-labeled phospholipids and membranes. J Am Chem Soc. 1971 Jan 27;93(2):314–326. doi: 10.1021/ja00731a005. [DOI] [PubMed] [Google Scholar]
  17. Jain M. K., Rogers J., Hendrickson H. S., Berg O. G. The chemical step is not rate-limiting during the hydrolysis by phospholipase A2 of mixed micelles of phospholipid and detergent. Biochemistry. 1993 Aug 17;32(32):8360–8367. doi: 10.1021/bi00083a040. [DOI] [PubMed] [Google Scholar]
  18. Janin J., Chothia C. The structure of protein-protein recognition sites. J Biol Chem. 1990 Sep 25;265(27):16027–16030. [PubMed] [Google Scholar]
  19. Lewis K. A., Bian J. R., Sweeney A., Roberts M. F. Asymmetric short-chain phosphatidylcholines: defining chain binding constraints in phospholipases. Biochemistry. 1990 Oct 23;29(42):9962–9970. doi: 10.1021/bi00494a029. [DOI] [PubMed] [Google Scholar]
  20. Lewis K. A., Soltys C. E., Yu K., Roberts M. F. Micellar bolaform and omega-carboxylate phosphatidylcholines as substrates for phospholipases. Biochemistry. 1994 May 3;33(17):5000–5010. doi: 10.1021/bi00183a002. [DOI] [PubMed] [Google Scholar]
  21. Liscovitch M., Cantley L. C. Lipid second messengers. Cell. 1994 May 6;77(3):329–334. doi: 10.1016/0092-8674(94)90148-1. [DOI] [PubMed] [Google Scholar]
  22. Moolenaar W. H. Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem. 1995 Jun 2;270(22):12949–12952. doi: 10.1074/jbc.270.22.12949. [DOI] [PubMed] [Google Scholar]
  23. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  24. Roberts M. F., Otnaess A. B., Kensil C. A., Dennis E. A. The specificity of phospholipase A2 and phospholipase C in a mixed micellar system. J Biol Chem. 1978 Feb 25;253(4):1252–1257. [PubMed] [Google Scholar]
  25. Schroit A. J., Madsen J. W. Synthesis and properties of radioiodinated phospholipid analogues that spontaneously undergo vesicle-vesicle and vesicle-cell transfer. Biochemistry. 1983 Jul 19;22(15):3617–3623. doi: 10.1021/bi00284a012. [DOI] [PubMed] [Google Scholar]
  26. Scott D. L., Otwinowski Z., Gelb M. H., Sigler P. B. Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue. Science. 1990 Dec 14;250(4987):1563–1566. doi: 10.1126/science.2274788. [DOI] [PubMed] [Google Scholar]
  27. Scott D. L., White S. P., Otwinowski Z., Yuan W., Gelb M. H., Sigler P. B. Interfacial catalysis: the mechanism of phospholipase A2. Science. 1990 Dec 14;250(4987):1541–1546. doi: 10.1126/science.2274785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Soltys C. E., Bian J., Roberts M. F. Polymerizable phosphatidylcholines: importance of phospholipid motions for optimum phospholipase A2 and C activity. Biochemistry. 1993 Sep 21;32(37):9545–9552. doi: 10.1021/bi00088a005. [DOI] [PubMed] [Google Scholar]
  29. Soltys C. E., Roberts M. F. Fluorescence studies of phosphatidylcholine micelle mixing: relevance to phospholipase kinetics. Biochemistry. 1994 Sep 27;33(38):11608–11617. doi: 10.1021/bi00204a023. [DOI] [PubMed] [Google Scholar]
  30. Spiegel S., Foster D., Kolesnick R. Signal transduction through lipid second messengers. Curr Opin Cell Biol. 1996 Apr;8(2):159–167. doi: 10.1016/s0955-0674(96)80061-5. [DOI] [PubMed] [Google Scholar]
  31. Waggoner A. S., Stryer L. Fluorescent probes of biological membranes. Proc Natl Acad Sci U S A. 1970 Oct;67(2):579–589. doi: 10.1073/pnas.67.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wells M. A. The mechanism of interfacial activation of phospholipase A2. Biochemistry. 1974 May 21;13(11):2248–2257. doi: 10.1021/bi00708a002. [DOI] [PubMed] [Google Scholar]
  33. White S. P., Scott D. L., Otwinowski Z., Gelb M. H., Sigler P. B. Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue. Science. 1990 Dec 14;250(4987):1560–1563. doi: 10.1126/science.2274787. [DOI] [PubMed] [Google Scholar]
  34. el-Sayed M. Y., DeBose C. D., Coury L. A., Roberts M. F. Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications. Biochim Biophys Acta. 1985 Dec 4;837(3):325–335. doi: 10.1016/0005-2760(85)90056-6. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES