Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.
Abstract: It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid (1-methylhistidine). Equine phospholipase was also modified by [13C]methyl p-nitrobenzenesulfonate and the methylated enzyme was studied by 13C NMR. The results indicate that the proton on the nitrogen in position 3 of the imidazole ring is involved in a strong interaction with a buried carboxylate group, thereby hindering rotation of the imidazole ring, and that the nitrogen in position 1 is involved in catalysis. These data are in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase. A catalytic mechanism is proposed in which a water molecule which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple acts as the nucleophile. A comparison is made between phospholipase A2 and the serine esterases.
Publication Date: 1980-02-19 PubMed ID: 7356955DOI: 10.1021/bi00545a021Google Scholar: Lookup
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Summary
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The research article explores the fundamental role of histidine-48 in the active centre of pancreatic phospholipase A2 by modifying the enzyme with different compounds, resulting in the loss of enzymatic activity. Detailed study of the methylated enzyme helped propose a catalytic mechanism and a comparison is made with serine esterases.
Understanding Histidine-48 in Pancreatic Phospholipase A2
- The researchers modified pancreatic phospholipase A2 enzyme with a number of bromo ketones and methyl benzenesulfonates to understand the role of histidine-48 as part of the enzyme’s active centre. This part of the research aimed to better understand the specific role this key amino acid plays in enzyme activity.
- It was found that rapid methylation occurred with methyl p-nitrobenzenesulfonate, resulting in a total loss of the enzymatic activity of the phospholipase. However, it was observed that the binding of the substrate and the cofactor calcium ions was not affected. Here, the researchers underline the crucial role of histidine-48 in maintaining the enzymatic function of pancreatic phospholipase A2.
Analysis of Methylated Equine Phospholipase
- Through amino acid analysis of methylated equine phospholipase, researchers found a loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid, also known as 1-methylhistidine. Thus it was clear that histidine-48 is important for the active state of pancreatic phospholipase A2.
- Further study of the methylated enzyme using [13C]methyl p-nitrobenzenesulfonate and 13C NMR showed a strong interaction between the proton on the nitrogen in position 3 of the imidazole ring and a buried carboxylate group, resulting in the hindering of the rotation of the imidazole ring.
- It was also observed that the nitrogen in position 1 was involved in the catalytic activity.
These observations were found to be in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase.
A Proposed Catalytic Mechanism
- Based on the research observations, a catalytic mechanism was proposed, in which a molecule of water, which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple, acts as the nucleophile. This proposed model offers an effective representation of the processes that are most likely happening at a molecular level.
- Finally, a comparison of phospholipase A2 with the serine esterases was also made, presumably to contextualize the findings within the broader landscape of enzymologic research. This can present potential similarities and differences in how these enzymes function, which can guide future research.
Cite This Article
APA
Verheij HM, Volwerk JJ, Jansen EH, Puyk WC, Dijkstra BW, Drenth J, de Haas GH.
(1980).
Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.
Biochemistry, 19(4), 743-750.
https://doi.org/10.1021/bi00545a021 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Benzenesulfonates / pharmacology
- Binding Sites
- Calcium / pharmacology
- Cattle
- Histidine
- Horses
- Ketones / pharmacology
- Kinetics
- Methylation
- Pancreas / enzymology
- Phospholipases / metabolism
- Phospholipases A / metabolism
- Phospholipases A2
- Protein Binding
- Spectrometry, Fluorescence
- Swine
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