Molecular Theory of the Catalytic Function of Phospholipase A2
| Project/Area Number |
02671022
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Osaka University of Pharmaceutical Science |
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Principal Investigator |
IKEDA Kiyoshi Osaka University of Pharmaceutical Science Professor, 薬学部, 教授 (50001053)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Phospholipase A2 / Calcium Ion / Kinetics / Phospholipid / Catalytic Function / Substrate Analog / ホスホリパ-ゼA_2 / pーブロムフェナシルブロミド / ランタニドイオン |
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| Research Abstract |
1. Kinetics of the hydrolysis of monodispersed and micellar sustrates, catalyzed by Group I or II phospholipase A_2 (PLA_2s) in the presence of a saturating Ca^<2+> concentration indicated that deprotonated state of the catalytic group His 48 and protonated state of Tyr 52, the site of which is located in close proximity to the imidazole ring of His 48 were found to be essential for the catalysis. The importance of an ionized state of the N-terminal alpha-amino group at the active site was also indicated for Group I enzymes. The pK values of both His 48 and Tyr 52 of the enzyme-Ca^<2+> complex shifted markedly to the alkaline side on binding to micellar substrates, whereas no significant pK shifts were noted for the bindings to monodispersed substrates.
2. Kinetic studies showed that Ca^<2+> binding to the both types of PLA_2s was essential for the catalysis. Substrate bindings to Group I PLA_2s were independent of the Ca^<2+> binding, whereas those for Group II enzymes were facilitated
… More
more than 10 times by the Ca^<2+> binging. The latter result was compatible with the hypothesis that an intermediate complex would be stabilized by coordination of the bound Ca^<2+> ion with the phosphoryl group and the carbonyl group at the sn-2 position of the substrate molecule. However, the former, the former result for Group I enzymes seemed incompatible for this mechanism. The X-ray crystallographic studies on the bindings of a substrate analog having an amide-bond instead of the sn-2 ester bond, dodecanoyl-2-aminohexanol-1-phosphoglycol, indicated that the carbonyl oxygen and phosphoryl moiety interact directly with the bound Ca^<2+> ion at the substrate binding site, suggesting that the binding of this analog should depend on the Ca^<2+> binding. Our studies in solution to confirm this showed the binding constants of this analog to both types of enzymes were increased significantly as the degrees of Ca^<2+> bindings increase, indicating that the structures of enzyme-substrate analog complex and enzyme-Ca^<2+>-analog complex in solution are very similar to each other as those in crystal and that the structures of the enzyme-analog complexes are stabilized by the Ca^<2+> binding. The enzyme-Ca^<2+>-genuine substrate complexes for the both types of PLA_2s are also considered to have similar structures.
3. Some metal ions including lantanide ions were showed to bind to PLA_2s and to their genuine-substrate complexes in a manner similar to that of Ca^<2+> ion and to produce the PLA_2 activites (less than 25%). Less
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Report
(4 results)
Research Products
(12 results)
