FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Biochemistry1980; 19(4); 743-750; doi: 10.1021/bi00545a021

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.
Get Full Text
Publication Date: 1980-02-19 PubMed ID: 7356955DOI: 10.1021/bi00545a021Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

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

Biochemistry
ISSN: 0006-2960
NlmUniqueID: 0370623
Country: United States
Language: English
Volume: 19
Issue: 4
Pages: 743-750

Researcher Affiliations

Verheij, H M
    Volwerk, J J
      Jansen, E H
        Puyk, W C
          Dijkstra, B W
            Drenth, J
              de Haas, G H

                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

                Citations

                This article has been cited 34 times.
                1. Castro-Amorim J, Novo de Oliveira A, Da Silva SL, Soares AM, Mukherjee AK, Ramos MJ, Fernandes PA. Catalytically Active Snake Venom PLA(2) Enzymes: An Overview of Its Elusive Mechanisms of Reaction. J Med Chem 2023 Apr 27;66(8):5364-5376.
                  doi: 10.1021/acs.jmedchem.3c00097pubmed: 37018514google scholar: lookup
                2. Guo X, Chen S, Cao J, Zhou J, Chen Y, Jamali MA, Zhang Y. Hydrolysis and oxidation of protein and lipids in dry-salted grass carp (Ctenopharyngodon idella) as affected by partial substitution of NaCl with KCl and amino acids. RSC Adv 2019 Dec 2;9(68):39545-39560.
                  doi: 10.1039/c9ra07019bpubmed: 35541390google scholar: lookup
                3. Matsui T, Kamata S, Ishii K, Maruno T, Ghanem N, Uchiyama S, Kato K, Suzuki A, Oda-Ueda N, Ogawa T, Tanaka Y. SDS-induced oligomerization of Lys49-phospholipase A(2) from snake venom. Sci Rep 2019 Feb 20;9(1):2330.
                  doi: 10.1038/s41598-019-38861-8pubmed: 30787342google scholar: lookup
                4. Zambelli VO, Chioato L, Gutierrez VP, Ward RJ, Cury Y. Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A(2). J Venom Anim Toxins Incl Trop Dis 2017;23:7.
                  doi: 10.1186/s40409-017-0099-6pubmed: 28203248google scholar: lookup
                5. Ximenes RM, Rabello MM, Araújo RM, Silveira ER, Fagundes FH, Diz-Filho EB, Buzzo SC, Soares VC, Toyama Dde O, Gaeta HH, Hernandes MZ, Monteiro HS, Toyama MH. Inhibition of Neurotoxic Secretory Phospholipases A(2) Enzymatic, Edematogenic, and Myotoxic Activities by Harpalycin 2, an Isoflavone Isolated from Harpalyce brasiliana Benth. Evid Based Complement Alternat Med 2012;2012:987517.
                  doi: 10.1155/2012/987517pubmed: 22899963google scholar: lookup
                6. Moon SH, Jenkins CM, Liu X, Guan S, Mancuso DJ, Gross RW. Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids. J Biol Chem 2012 Apr 27;287(18):14880-95.
                  doi: 10.1074/jbc.M111.336776pubmed: 22389508google scholar: lookup
                7. Burke JE, Dennis EA. Phospholipase A2 biochemistry. Cardiovasc Drugs Ther 2009 Feb;23(1):49-59.
                  doi: 10.1007/s10557-008-6132-9pubmed: 18931897google scholar: lookup
                8. Zhou X, Tan TC, Valiyaveettil S, Go ML, Kini RM, Velazquez-Campoy A, Sivaraman J. Structural characterization of myotoxic ecarpholin S from Echis carinatus venom. Biophys J 2008 Oct;95(7):3366-80.
                  doi: 10.1529/biophysj.107.117747pubmed: 18586854google scholar: lookup
                9. Wee CL, Balali-Mood K, Gavaghan D, Sansom MS. The interaction of phospholipase A2 with a phospholipid bilayer: coarse-grained molecular dynamics simulations. Biophys J 2008 Aug;95(4):1649-57.
                  doi: 10.1529/biophysj.107.123190pubmed: 18469074google scholar: lookup
                10. Singh G, Gourinath S, Saravanan K, Sharma S, Bhanumathi S, Betzel Ch, Srinivasan A, Singh TP. Sequence-induced trimerization of phospholipase A2: structure of a trimeric isoform of PLA2 from common krait (Bungarus caeruleus) at 2.5 A resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun 2005 Jan 1;61(Pt 1):8-13.
                  doi: 10.1107/S1744309104025503pubmed: 16508078google scholar: lookup
                11. Sekar K, Rajakannan V, Gayathri D, Velmurugan D, Poi MJ, Dauter M, Dauter Z, Tsai MD. Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2. Acta Crystallogr Sect F Struct Biol Cryst Commun 2005 Jan 1;61(Pt 1):3-7.
                  doi: 10.1107/S1744309104021748pubmed: 16508077google scholar: lookup
                12. Toyama MH, Toyama DO, Joazeiro PP, Carneiro EM, Beriam LO, Marangoni LS, Boschero AC. Biological and structural characterization of a new PLA2 from the Crotalus durissus collilineatus venom. Protein J 2005 Feb;24(2):103-12.
                  doi: 10.1007/s10930-004-1517-5pubmed: 16003952google scholar: lookup
                13. Singh G, Jasti J, Saravanan K, Sharma S, Kaur P, Srinivasan A, Singh TP. Crystal structure of the complex formed between a group I phospholipase A2 and a naturally occurring fatty acid at 2.7 A resolution. Protein Sci 2005 Feb;14(2):395-400.
                  doi: 10.1110/ps.041115505pubmed: 15659372google scholar: lookup
                14. Warwicker J. Improved pKa calculations through flexibility based sampling of a water-dominated interaction scheme. Protein Sci 2004 Oct;13(10):2793-805.
                  doi: 10.1110/ps.04785604pubmed: 15388865google scholar: lookup
                15. Sá JM, Chioato L, Ferreira TL, De Oliveira AH, Ruller R, Rosa JC, Greene LJ, Ward RJ. Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity. Biochem J 2004 Aug 15;382(Pt 1):191-8.
                  doi: 10.1042/BJ20031946pubmed: 15147240google scholar: lookup
                16. Tatulian SA. Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites. Biophys J 2003 Mar;84(3):1773-83.
                  doi: 10.1016/S0006-3495(03)74985-6pubmed: 12609879google scholar: lookup
                17. Ward RJ, Chioato L, de Oliveira AH, Ruller R, Sá JM. Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis. Biochem J 2002 Feb 15;362(Pt 1):89-96.
                  doi: 10.1042/0264-6021:3620089pubmed: 11829743google scholar: lookup
                18. Chang LS, Wen EY, Chang CC. The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2. J Protein Chem 1996 Apr;15(3):255-60.
                  doi: 10.1007/BF01887113pubmed: 8804572google scholar: lookup
                19. Symer DE, Paznekas WA, Shin HS. A requirement for membrane-associated phospholipase A2 in platelet cytotoxicity activated by receptors for immunoglobulin G and complement. J Exp Med 1993 Apr 1;177(4):937-47.
                  doi: 10.1084/jem.177.4.937pubmed: 8459221google scholar: lookup
                20. Weng X, Luecke H, Song IS, Kang DS, Kim SH, Huber R. Crystal structure of human annexin I at 2.5 A resolution. Protein Sci 1993 Mar;2(3):448-58.
                  doi: 10.1002/pro.5560020317pubmed: 8453382google scholar: lookup
                21. Lewendon A, Shaw WV. The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase. Biochem J 1993 Feb 15;290 ( Pt 1)(Pt 1):15-9.
                  doi: 10.1042/bj2900015pubmed: 8439283google scholar: lookup
                22. Rehfeldt W, Resch K, Goppelt-Struebe M. Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca(2+)-dependent membrane association. Biochem J 1993 Jul 1;293 ( Pt 1)(Pt 1):255-61.
                  doi: 10.1042/bj2930255pubmed: 8328965google scholar: lookup
                23. van den Berg B, Tessari M, Boelens R, Dijkman R, Kaptein R, de Haas GH, Verheij HM. Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor. J Biomol NMR 1995 Feb;5(2):110-21.
                  doi: 10.1007/BF00208802pubmed: 7703697google scholar: lookup
                24. Buhl WJ, Eisenlohr LM, Preuss I, Gehring U. A novel phospholipase A2 from human placenta. Biochem J 1995 Oct 1;311 ( Pt 1)(Pt 1):147-53.
                  doi: 10.1042/bj3110147pubmed: 7575446google scholar: lookup
                25. Chu ST, Chen YH. The intrinsic tryptophan fluorescence of beta 1-bungarotoxin and the Ca2+-binding domains of the toxin as probed with Tb3+ luminescence. Biochem J 1989 Sep 15;262(3):773-9.
                  doi: 10.1042/bj2620773pubmed: 2590165google scholar: lookup
                26. Scott DL, White SP, Otwinowski Z, Yuan W, Gelb MH, Sigler PB. Interfacial catalysis: the mechanism of phospholipase A2. Science 1990 Dec 14;250(4987):1541-6.
                  doi: 10.1126/science.2274785pubmed: 2274785google scholar: lookup
                27. Rogers MV, Henkle KJ, Herrmann V, McLaren DJ, Mitchell GF. Evidence that a 16-kilodalton integral membrane protein antigen from Schistosoma japonicum adult worms is a type A2 phospholipase. Infect Immun 1991 Apr;59(4):1442-7.
                  doi: 10.1128/iai.59.4.1442-1447.1991pubmed: 2004822google scholar: lookup
                28. Yu L, Dennis EA. Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues. Proc Natl Acad Sci U S A 1991 Oct 15;88(20):9325-9.
                  doi: 10.1073/pnas.88.20.9325pubmed: 1924395google scholar: lookup
                29. Slaich PK, Primrose WU, Robinson DH, Wharton CW, White AJ, Drabble K, Roberts GC. The binding of amide substrate analogues to phospholipase A2. Studies by 13C-nuclear-magnetic-resonance and infrared spectroscopy. Biochem J 1992 Nov 15;288 ( Pt 1)(Pt 1):167-73.
                  doi: 10.1042/bj2880167pubmed: 1445261google scholar: lookup
                30. Wen H, Huang H, Zhao Y, Sun J, Zhang Y. The effects of Lactobacillus plantarum and Staphylococcus simulans on the physicochemical properties, colony composition and flavor characteristics of low-sodium dry cured goose. Poult Sci 2026 Feb;105(2):106221.
                  doi: 10.1016/j.psj.2025.106221pubmed: 41391362google scholar: lookup
                31. Muntean M, Florea A. Phospholipase A2-A Significant Bio-Active Molecule in Honeybee (Apis mellifera L.) Venom. Molecules 2025 Jun 17;30(12).
                  doi: 10.3390/molecules30122623pubmed: 40572586google scholar: lookup
                32. Alves AEF, Formiga ALD, Uchoa AFC, Cardoso ALMR, Rodrigues EOAL, de Araujo Pereira GM, de Padua Farias Bezerra Leite J, da Silva LFA, de Sousa NF, da Silva Sobral M, Scotti MT, Scotti L, Xavier Junior FH. Targets Involved in the Pharmacology of Bothrops Snakebite: Statu Quo and Future Perspectives. Curr Drug Targets 2025;26(7):454-469.
                  doi: 10.2174/0113894501352925250225045555pubmed: 40017248google scholar: lookup
                33. Castro-Amorim J, Pinto AV, Mukherjee AK, Ramos MJ, Fernandes PA. Beyond Fang's fury: a computational study of the enzyme-membrane interaction and catalytic pathway of the snake venom phospholipase A(2) toxin. Chem Sci 2025 Jan 22;16(4):1974-1985.
                  doi: 10.1039/d4sc06511epubmed: 39759936google scholar: lookup
                34. Srinivasan K, Nampoothiri M, Khandibharad S, Singh S, Nayak AG, Hariharapura RC. Proteomic diversity of Russell's viper venom: exploring PLA2 isoforms, pharmacological effects, and inhibitory approaches. Arch Toxicol 2024 Nov;98(11):3569-3584.
                  doi: 10.1007/s00204-024-03849-5pubmed: 39181947google scholar: lookup
                Subscribe to our newsletter
                Join 100,151 horse owners like you who receive our email updates on horse health and nutrition.