Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study

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Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study. / da Graça Thrige, D; Buur, J R; Jørgensen, Flemming Steen.

In: Biopolymers, Vol. 42, No. 3, 1997, p. 319-36.

Research output: Contribution to journalJournal articleResearchpeer-review

Harvard

da Graça Thrige, D, Buur, JR & Jørgensen, FS 1997, 'Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study', Biopolymers, vol. 42, no. 3, pp. 319-36. https://doi.org/10.1002/(SICI)1097-0282(199709)42:3<319::aid-bip5>3.0.CO;2-P

APA

da Graça Thrige, D., Buur, J. R., & Jørgensen, F. S. (1997). Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study. Biopolymers, 42(3), 319-36. https://doi.org/10.1002/(SICI)1097-0282(199709)42:3<319::aid-bip5>3.0.CO;2-P

Vancouver

da Graça Thrige D, Buur JR, Jørgensen FS. Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study. Biopolymers. 1997;42(3):319-36. https://doi.org/10.1002/(SICI)1097-0282(199709)42:3<319::aid-bip5>3.0.CO;2-P

Author

da Graça Thrige, D ; Buur, J R ; Jørgensen, Flemming Steen. / Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study. In: Biopolymers. 1997 ; Vol. 42, No. 3. pp. 319-36.

Bibtex

@article{668ade9bbda54d36a748b4a93112f62f,
title = "Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study",
abstract = "For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.",
keywords = "Bacillus cereus, Binding Sites, Computer Simulation, Crystallography, X-Ray, Hydrogen Bonding, Models, Molecular, Phospholipids, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Type C Phospholipases, Zinc",
author = "{da Gra{\cc}a Thrige}, D and Buur, {J R} and J{\o}rgensen, {Flemming Steen}",
year = "1997",
doi = "10.1002/(SICI)1097-0282(199709)42:3<319::aid-bip5>3.0.CO;2-P",
language = "English",
volume = "42",
pages = "319--36",
journal = "Biopolymers",
issn = "0006-3525",
publisher = "Wiley",
number = "3",

}

RIS

TY - JOUR

T1 - Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus. a molecular mechanics and molecular dynamics study

AU - da Graça Thrige, D

AU - Buur, J R

AU - Jørgensen, Flemming Steen

PY - 1997

Y1 - 1997

N2 - For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.

AB - For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.

KW - Bacillus cereus

KW - Binding Sites

KW - Computer Simulation

KW - Crystallography, X-Ray

KW - Hydrogen Bonding

KW - Models, Molecular

KW - Phospholipids

KW - Protein Binding

KW - Protein Conformation

KW - Protein Structure, Tertiary

KW - Type C Phospholipases

KW - Zinc

U2 - 10.1002/(SICI)1097-0282(199709)42:3<319::aid-bip5>3.0.CO;2-P

DO - 10.1002/(SICI)1097-0282(199709)42:3<319::aid-bip5>3.0.CO;2-P

M3 - Journal article

C2 - 9279125

VL - 42

SP - 319

EP - 336

JO - Biopolymers

JF - Biopolymers

SN - 0006-3525

IS - 3

ER -

ID: 38394473