Localization of the second calcium ion binding site in porcine and equine phospholipase A2.
Abstract: At alkaline pH porcine pancreatic phospholipase A2 is known to bind two Ca2+ ions per protein molecule. One Ca2+ ion is strongly bound to the active site and is essential for enzyme activity. A second Ca2+ ion binds more weakly to the protein and improves the affinity of the enzyme for lipid-water interfaces severalfold at high pH values. A group having a pK around 6 controls enzyme binding to lipid-water interfaces in the absence of Ca2+. By use of proton titration techniques this group is now identified to be a carboxylate having an abnormally high pK. Its pK shifts to a value around 4.5 in the presence of high Ca2+ concentrations, suggesting that the carboxylate is involved in binding the second Ca2+ ion. The carboxylate was identified to be Glu71 by comparing proton titration experiments on porcine pancreatic phospholipase A2 and an isoenzyme. The isoenzyme differs by only four residues from the most abundant enzyme, lacking the carboxylate at position 71 (Asn for Glu). The isoenzyme also appeared to be devoid of an abnormal carboxylate. Identification of Glu71 as the abnormal carboxylate in the porcine enzyme was substantiated by comparison with enzymes from other sources. Kinetic experiments on the various phospholipases finally demonstrated that enzyme species containing Glu71 bind a second Ca2+ ion to the low-affinity site, whereas enzymes lacking Glu71 also lack this second site. These experiments confirm the suggestion that Glu71 is one of the ligands for Ca2+ in the low-affinity site.
Publication Date: 1983-05-10 PubMed ID: 6860643DOI: 10.1021/bi00279a025Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article discusses the identification of a second calcium ion binding site in porcine and equine phospholipase A2 enzymes. This site, associated with the presence of a specific carboxylate group (Glu71), enhances the enzyme’s affinity for lipid-water interfaces at high pH values.
Overview of study
- The study aimed to determine the location of the second calcium ion binding site in porcine and equine phospholipase A2.
- It is established that at alkaline pH, porcine pancreatic phospholipase A2 can bind two calcium ions per protein molecule. One of these is strongly bound to the active site and is crucial for enzyme activity. A second calcium ion binds more weakly, thereby increasing the enzyme’s affinity for lipid-water interfaces at higher pH values.
A novel carboxylate group identified
- A group with a pK around 6 – a unit used to measure the acidity or alkalinity of a solution – controls the enzyme binding to lipid-water interfaces in the absence of calcium ions.
- Through the use of proton titration techniques, the researchers identified this group as a carboxylate, which has an abnormally high pK. This carboxylate behaves atypically by shifting its pK value near 4.5 when present in high concentrations of calcium ions.
- This suggests that the carboxylate may have a role in binding the second calcium ion.
The role of Glu71
- The abnormal carboxylate was found to be Glu71, identified through comparison of the porcine pancreatic phospholipase A2 with an isoenzyme. The isoenzyme differed by only four residue positions, one being the absence of the carboxylate at position 71, replaced by Asn for Glu.
- The enzymes that contain Glu71 bind a second calcium ion to their low-affinity site. On the other hand, enzymes that lack Glu71 also lack this second site. This demonstrates Glu71’s role in calcium ion binding.
Conclusion
- This research concludes by suggesting that Glu71 is one of the ligands for calcium in the low-affinity site of the phospholipase A2 enzyme.
- The findings could have implications on our understanding of enzyme’s activity and its interaction with calcium ions.
Cite This Article
APA
Donné-Op den Kelder GM, de Haas GH, Egmond MR.
(1983).
Localization of the second calcium ion binding site in porcine and equine phospholipase A2.
Biochemistry, 22(10), 2470-2478.
https://doi.org/10.1021/bi00279a025 Publication
Researcher Affiliations
MeSH Terms
- Amino Acid Sequence
- Amino Acids / analysis
- Animals
- Binding Sites
- Calcium / metabolism
- Cattle
- Horses
- Humans
- Kinetics
- Mathematics
- Pancreas / enzymology
- Phospholipases / metabolism
- Phospholipases A / metabolism
- Phospholipases A2
- Protein Binding
- Protein Conformation
- Species Specificity
- Swine
Citations
This article has been cited 2 times.- Mezna M, Ahmad T, Chettibi S, Drainas D, Lawrence AJ. Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms. Biochem J 1994 Jul 15;301 ( Pt 2)(Pt 2):503-8.
- Fujii S, Tahara Y, Toyomoto M, Hada S, Nishimura H, Inoue S, Ikeda K, Inagaki Y, Katsumura S, Samejima Y. Chemical modification and inactivation of phospholipases A2 by a manoalide analogue. Biochem J 1995 May 15;308 ( Pt 1)(Pt 1):297-304.
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