1bwp
From Proteopedia
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==Overview== | ==Overview== | ||
| - | Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s | + | Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Derewenda, U.]] | [[Category: Derewenda, U.]] | ||
[[Category: Derewenda, Z.]] | [[Category: Derewenda, Z.]] | ||
| - | [[Category: Ho, Y | + | [[Category: Ho, Y S.]] |
[[Category: Inoue, K.]] | [[Category: Inoue, K.]] | ||
[[Category: Li, J.]] | [[Category: Li, J.]] | ||
[[Category: Masuyama, J.]] | [[Category: Masuyama, J.]] | ||
| - | [[Category: Sheffield, P | + | [[Category: Sheffield, P J.]] |
[[Category: acetylhydrolase hydrolase]] | [[Category: acetylhydrolase hydrolase]] | ||
[[Category: lipid degradation]] | [[Category: lipid degradation]] | ||
[[Category: platelet factor]] | [[Category: platelet factor]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:59:52 2008'' |
Revision as of 09:59, 21 February 2008
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PROBING THE SUBSTRATE SPECIFICITY OF THE INTRACELLULAR BRAIN PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE
Overview
Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.
About this Structure
1BWP is a Single protein structure of sequence from Bos taurus. Active as 1-alkyl-2-acetylglycerophosphocholine esterase, with EC number 3.1.1.47 Known structural/functional Site: . Full crystallographic information is available from OCA.
Reference
Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase., Ho YS, Sheffield PJ, Masuyama J, Arai H, Li J, Aoki J, Inoue K, Derewenda U, Derewenda ZS, Protein Eng. 1999 Aug;12(8):693-700. PMID:10469831
Page seeded by OCA on Thu Feb 21 11:59:52 2008
