Abstract
To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.
Full Text
The Full Text of this article is available as a PDF (6.5 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aguiar A., De Haas G. H., Jansen E. H., Slotboom A. J., Williams R. J. Proton-nuclear-magnetic-resonance/pH-titration studies of the histidines of pancreatic phospholipase A2. Eur J Biochem. 1979 Oct 15;100(2):511–518. doi: 10.1111/j.1432-1033.1979.tb04196.x. [DOI] [PubMed] [Google Scholar]
- Berg O. G., Yu B. Z., Rogers J., Jain M. K. Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants. Biochemistry. 1991 Jul 23;30(29):7283–7297. doi: 10.1021/bi00243a034. [DOI] [PubMed] [Google Scholar]
- Brunie S., Bolin J., Gewirth D., Sigler P. B. The refined crystal structure of dimeric phospholipase A2 at 2.5 A. Access to a shielded catalytic center. J Biol Chem. 1985 Aug 15;260(17):9742–9749. [PubMed] [Google Scholar]
- Dijkstra B. W., Kalk K. H., Drenth J., de Haas G. H., Egmond M. R., Slotboom A. J. Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2. Biochemistry. 1984 Jun 5;23(12):2759–2766. doi: 10.1021/bi00307a035. [DOI] [PubMed] [Google Scholar]
- Gean P. W., Huang C. C., Kuo J. R., Lin J. H., Yi P. L., Tsai J. J. Analysis of carbamazepine's anticonvulsant actions in hippocampal and amygdaloid slices of the rat. Chin J Physiol. 1993;36(4):199–204. [PubMed] [Google Scholar]
- Jain M. K., Gelb M. H. Phospholipase A2-catalyzed hydrolysis of vesicles: uses of interfacial catalysis in the scooting mode. Methods Enzymol. 1991;197:112–125. doi: 10.1016/0076-6879(91)97138-o. [DOI] [PubMed] [Google Scholar]
- Jain M. K., Maliwal B. P. Spectroscopic properties of the states of pig pancreatic phospholipase A2 at interfaces and their possible molecular origin. Biochemistry. 1993 Nov 9;32(44):11838–11846. doi: 10.1021/bi00095a012. [DOI] [PubMed] [Google Scholar]
- Jones T. A. Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. Methods Enzymol. 1985;115:157–171. doi: 10.1016/0076-6879(85)15014-7. [DOI] [PubMed] [Google Scholar]
- Noel J. P., Bingman C. A., Deng T. L., Dupureur C. M., Hamilton K. J., Jiang R. T., Kwak J. G., Sekharudu C., Sundaralingam M., Tsai M. D. Phospholipase A2 engineering. X-ray structural and functional evidence for the interaction of lysine-56 with substrates. Biochemistry. 1991 Dec 24;30(51):11801–11811. doi: 10.1021/bi00115a010. [DOI] [PubMed] [Google Scholar]
- Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Sekharudu C., Ramakrishnan B., Huang B., Jiang R. T., Dupureur C. M., Tsai M. D., Sundaralingam M. Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines. Protein Sci. 1992 Dec;1(12):1585–1594. doi: 10.1002/pro.5560011206. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Slotboom A. J., van Dam-Mieras M. C., de Haas G. H. Regulation of phospholipase A2 activity by different lipid-water interfaces. J Biol Chem. 1977 May 10;252(9):2948–2951. [PubMed] [Google Scholar]