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Studies on Active site Mutants of Human Non-Pancreatic secreted Phospholipase A2

Studies on Active site Mutants of Human Non-Pancreatic secreted Phospholipase A2
Studies on Active site Mutants of Human Non-Pancreatic secreted Phospholipase A2

Phospholipase A2 (PLA2) is an enzyme that catalyses the hydrolysis of phospholipids at the sn-2 fatty-acyl bond, liberating free fatty acids and lysophospholipids. As many as eleven different forms of the enzyme have been characterised, including both secreted and cytosolic, calcium dependent and independent varieties. These PLA2S are involved in a range of physiological processes including phospholipid digestion, signal transduction and host defence, and have been isolated from a diverse range of organisms and cells.

This thesis is concerned with the human non-pancreatic secreted form of PLA2 (hnpsPLA2), which is secreted by a variety of cells including platelets, macrophages and smooth muscle cells. HnpsPLA2 is a 14 kDa, calcium-dependent extracellular enzyme believed to be an acute phase protein, with an important antimicrobial role. It is the binding of this enzyme to the phospholipid interface that is crucial in the physiological regulation of the enzyme. In order to study interfacial binding in the absence of catalysis, structurally intact but inactive mutants were required. The production of the mutants H48Q, H48N and H48A appeared to be the obvious strategy based on previous work on other secreted phospholipases A2 from bovine pancreas and bee venom. However, the mutant H48Q unexpectedly retained significant (~4%) catalytic activity that was contrary to all previous work in the field.

In this thesis it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions. The crystallisation of the H48Q mutant to a resolution of 1.5 Å allowed a comparison with the corresponding wild-type enzyme (N1A) that was also crystallised. This comparison revealed that all the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins and the two catalytic water molecules were clearly resolved in the H48Q mutant. A 'two-water mechanism' in which the second water molecule at the active site hydrogen bonds to the glutamine could explain the residual activity of the H48Q. A second active site mutant (H48N) showed minimal activity and was used to study the interfacial binding properties of the enzyme. The results highlighted the use of this mutant in interfacial binding assays and confirmed the high affinity of this enzyme for anionic interfaces. The third active site mutant, H48A, was completely inactive. However, this mutant could not be expressed in significant amounts and was not further investigated.

University of Southampton
Edwards, Suzanne H
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Edwards, Suzanne H
1ccc09dd-8d98-47d6-9ef6-6233d5227da7

Edwards, Suzanne H (2001) Studies on Active site Mutants of Human Non-Pancreatic secreted Phospholipase A2. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Phospholipase A2 (PLA2) is an enzyme that catalyses the hydrolysis of phospholipids at the sn-2 fatty-acyl bond, liberating free fatty acids and lysophospholipids. As many as eleven different forms of the enzyme have been characterised, including both secreted and cytosolic, calcium dependent and independent varieties. These PLA2S are involved in a range of physiological processes including phospholipid digestion, signal transduction and host defence, and have been isolated from a diverse range of organisms and cells.

This thesis is concerned with the human non-pancreatic secreted form of PLA2 (hnpsPLA2), which is secreted by a variety of cells including platelets, macrophages and smooth muscle cells. HnpsPLA2 is a 14 kDa, calcium-dependent extracellular enzyme believed to be an acute phase protein, with an important antimicrobial role. It is the binding of this enzyme to the phospholipid interface that is crucial in the physiological regulation of the enzyme. In order to study interfacial binding in the absence of catalysis, structurally intact but inactive mutants were required. The production of the mutants H48Q, H48N and H48A appeared to be the obvious strategy based on previous work on other secreted phospholipases A2 from bovine pancreas and bee venom. However, the mutant H48Q unexpectedly retained significant (~4%) catalytic activity that was contrary to all previous work in the field.

In this thesis it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions. The crystallisation of the H48Q mutant to a resolution of 1.5 Å allowed a comparison with the corresponding wild-type enzyme (N1A) that was also crystallised. This comparison revealed that all the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins and the two catalytic water molecules were clearly resolved in the H48Q mutant. A 'two-water mechanism' in which the second water molecule at the active site hydrogen bonds to the glutamine could explain the residual activity of the H48Q. A second active site mutant (H48N) showed minimal activity and was used to study the interfacial binding properties of the enzyme. The results highlighted the use of this mutant in interfacial binding assays and confirmed the high affinity of this enzyme for anionic interfaces. The third active site mutant, H48A, was completely inactive. However, this mutant could not be expressed in significant amounts and was not further investigated.

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Published date: 2001

Identifiers

Local EPrints ID: 464512
URI: http://eprints.soton.ac.uk/id/eprint/464512
PURE UUID: fc115a75-49d8-4402-bd30-94645eb3b7f6

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Date deposited: 04 Jul 2022 23:43
Last modified: 05 Jul 2022 02:05

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Author: Suzanne H Edwards

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