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The importance of glycine-30 for enzymatic activity of phospholipase A2
A C Bekkers1, P A Franken, E Toxopeus
1Department of Enzymology and Protein Engineering, University of Utrecht, CBLE, University Centre De Uithof, The Netherlands.
Abstract:
The nearly conserved glycine-30 in porcine pancreatic phospholipase A2 has been replaced by serine. The resulting mutant G30S was expressed in Escherichia coli, purified and characterized. The mutation caused a significant drop in enzymatic activity towards monomeric and aggregated substrates, but had a limited effect on substrate binding. In contrast the affinity for calcium ions, the essential cofactor, was reduced 10-fold. The reduced enzymatic activity is attributed to a reduced stabilization of the transition state. The results are discussed in view of naturally occurring inactive phospholipase A2 homologues from snake venom.
Insights
Replacing glycine-30 with serine in porcine pancreatic phospholipase A2 (G30S mutant) significantly reduced enzyme activity and calcium ion affinity. This suggests altered transition state stabilization, offering insights into inactive phospholipase A2 homologues.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Porcine pancreatic phospholipase A2 is a key enzyme in lipid metabolism.
- The glycine at position 30 is highly conserved across species.
- Understanding the role of conserved residues is crucial for enzyme function studies.
Purpose of the Study:
- To investigate the functional impact of substituting glycine-30 with serine in porcine pancreatic phospholipase A2.
- To characterize the enzymatic activity, substrate binding, and cofactor affinity of the G30S mutant.
- To elucidate the structural and mechanistic basis for changes in enzyme activity.
Main Methods:
- Site-directed mutagenesis to create the G30S mutant.
- Expression and purification of the mutant enzyme in Escherichia coli.
- Enzymatic assays using monomeric and aggregated substrates.
- Determination of substrate binding affinity and calcium ion affinity.
Main Results:
- The G30S mutation led to a significant decrease in enzymatic activity against both monomeric and aggregated substrates.
- Substrate binding affinity was only marginally affected by the mutation.
- A 10-fold reduction in calcium ion affinity was observed for the G30S mutant.
- The reduced activity is likely due to impaired stabilization of the transition state.
Conclusions:
- The glycine-30 residue plays a critical role in maintaining the catalytic efficiency and calcium ion binding of porcine pancreatic phospholipase A2.
- The G30S mutation affects transition state stabilization, impacting overall enzymatic function.
- These findings provide insights into the mechanisms of inactive phospholipase A2 homologues found in snake venom.