Binding mechanism of spinosine and venenatine molecules with p300 HAT enzyme: Molecular screening,molecular dynamics and free-energy analysis

The chromatin modification is regulated by the histone acetyltransferase (HAT) and histone deacetyltransferase (HDAC) enzymes; abnormal function of these enzymes leads to several malignant diseases. The inhibition of these enzymes using natural ligand molecules is an emerging technique to cure these diseases. The in vitro analysis of natural molecules, venenatine, spinosine, palmatine and taxodione are giving the best inhibition rate against p300 HAT enzyme. However, the detailed understanding of binding and the stability of these molecules with p300 HAT is not yet known. The aim of the present study is focused to determine the binding strength of the molecules from molecular dynamics simulation analysis. The docking analysis confirms that, the venenatine (−6.97 kcal/mol - conformer 8), spinosine (−6.52 kcal/mol conformer −10), palmatine (−5.72 kcal/mol conformer-3) and taxodione (−4.99 kcal/mol conformer-4) molecules form strong hydrogen bonding interactions with the key amino acid residues (Arg1410, Thr1411 and Trp1466) present in the active site of p300. In the molecular dynamics (MD) simulation, the spinosine retain these key interactions with the active site amino acid residues (Arg1410, Thr1411, and Trp1466) than venenatine and are stable throughout the simulation. The RMSD value of spinosine (0.5 to 1.3 Å) and venenatine (0.3 to 1.3 Å) are almost equal during the MD simulation. However, during the MD simulation, the intermolecular interaction between venenatine and the active site amino acid residues (Arg1410, Thr1411, and Trp1466) decreased on comparing with the spinosine-p300 interaction. The binding free energy of the spinosine (−15.30 kcal/mol) is relatively higher than the venenatine (−11.8 kcal/mol); this increment is attributed to the strong hydrogen bonding interactions of spinosine molecule with the active site amino acid residues of p300.     

Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking,molecular dynamics,and charge density analysis

Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer’s disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.     

Kinetic and mass spectrometric analysis of p300 histone acetyltransferase domain autoacetylation

Acetylation of proteins by p300 histone acetyltransferase plays a critical role in the regulation of gene expression. The prior discovery of an autoacetylated regulatory loop in the p300 histone acetyltransferase (HAT) domain prompted us to further explore the mechanisms of p300 autoacetylation. Here we have described a kinetic and mass spectrometric analysis of p300 HAT autoacetylation. The rate of p300 HAT autoacetylation was approximately fourth order with respect to p300 HAT domain concentration and thus appeared to be a highly cooperative process. By showing that a catalytically defective p300 HAT domain could be efficiently acetylated by active p300 HAT, we deduced that autoacetylation occurs primarily by an intermolecular mechanism. This was further confirmed using a semisynthetic biotinylated p300 HAT domain that could be physically separated from the catalytically defective p300 HAT by avidin affinity chromatography. Autoacetylation catalyzed by p300 HAT was approximately 1000-fold more efficient than PCAF (p300/CREB-binding protein-associated factor)-mediated acetylation of catalytically defective p300 HAT. Using a novel tandem mass spectrometric approach, it was found to be possible to observe up to 17 autoacetylation events within the intact p300 regulatory loop. Kinetic analysis of the site specificity of p300 autoacetylation reveals a class of rapid events followed by a slower set of modifications. Several of these rapid autoacetylation sites correlate with an acetyltransferase-activating function based on prior mutagenesis analysis.     

Investigation of intermolecular interactions and stability of verubecestat in the active site of BACE1: Development of first model from QM/MM-based charge density and MD analysis

Alzheimer disease (AD) is a cruel neurodegenerative disorder caused by the deposition of amyloid β (Aβ) peptide inside the brain. The β-secretase (beta amyloid precursor protein (APP) cleaving enzyme 1, BACE1) is one of the enzymes involved in the cleavage of APP that leads to the Aβ formation and it is the primary target for the treatment of AD. Recent report outlines that verubecestat molecule strongly inhibits BACE1; however, its structure, binding mechanism and the stability in the active site of BACE1 are not yet known. The present study aims to determine the structure, binding affinity and the stability of verubecestat molecule in the active site of BACE1 from the molecular docking, quantum mechanics/molecular mechanics (QM/MM)-based charge density analysis and molecular dynamics simulation. Verubecestat molecule was docked at BACE1; it shows high binding affinity towards BACE1. Further, the conformational geometry and the intermolecular interactions of verubecestat in the active site of BACE1 were determined. The molecule forms strong interaction with the neighboring amino acids in the active site of BACE1. The onsite QM/MM-based charge density analysis reveals the nature of charge density distribution and the topological properties of intermolecular interactions of verubecestat molecule in the active site of BACE1. The calculated electrostatic potential (ESP) of verubecestat in the active site of BACE1 displays high negative and positive ESP regions of the molecule. This onsite QM/MM analysis is more relevant to the physiological situation. The molecular dynamics simulation has been performed, which confirms the high stability and compactness of verubecestat in the active site of BACE1. The MM-generalized Born surface area and MM-Poisson Boltzmann surface area free energy calculations of verubecestat–BACE1 also confirm the high binding affinity of verubecestat.

Communicated by Ramaswamy H. Sarma     

Discovery of new potential triplet acting inhibitor for Alzheimer’s disease via X-ray crystallography,molecular docking and molecular dynamics

Abstract

The most common brain disorder of late life is Alzheimer’s disease (AD), which is highly complicating dementia. There are several drug targets which are reported to control the severe level of AD; notably, acetylcholinesterase, β-Secretase and glycogen synthase kinase enzymes are approached as a good drug targets for AD. Hence, the present study mainly focused to discover newly synthesized molecule (7-propyl-6H-pyrano[3,2-c:5,6-c']dichromene-6,8(7H)-dione) as a potential triplet acting drug for above said enzymes through the analysis of X-ray crystallography, molecular docking, molecular dynamics and quantum chemical calculation. The target drug molecule was crystallized in the monoclinic crystal structure with P21/n space group. The structure was solved by SHELXS and refined by SHELXL. The crystal packing is stabilized by C???H···O type of interactions. Further, the induced fit docking shows that the molecule has high docking score, glide energy, favorable hydrogen bonding and hydrophobic interactions on the protein targets. The molecular dynamics simulation was performed to understand the stability of the molecule in the presence of active site environment. Finally, quantum chemical calculation has been carried out for the molecule in gas phase and for the corresponding molecule lifted from the active site region. The structural comparison between gas phase and active site helps to understand the conformational modification of the molecule in the active site.

Communicated by Ramaswamy H. Sarma     

Towards an understanding of the arginine-aspartate interaction.

We have made a comparison of the geometries of intra- and intermolecular arginine-aspartate interactions by extracting orientation information from protein co-ordinate data. The results show a pronounced difference, with both types of interaction preferring to form twin N-H . . . O = C hydrogen bonds, but involving different nitrogen atoms. In intramolecular interactions, the aspartate favours a "side on" geometry, forming hydrogen bonds with N epsilon and N eta 2; in the intermolecular case, however, "end on" contacts involving N eta 1 and N eta 2 of the arginine are preferred. We have used Distributed Multipole Analysis of the methylguanidinium-acetate system to model the electrostatic component of the arginine-aspartate ion pair interaction in vacuo. We find, in agreement with the experimental arginine-aspartate distribution, that side on and end on doubly N-H . . . O = C hydrogen-bonded configurations are clearly the most favourable, with the side on being marginally lower in energy. Thus, despite the many competing side-chain interactions in proteins, many arginine-aspartate pairs adopt one of the minimum electrostatic energy conformations, or one close to a minimum. Within each of the two regions (side on and end on) we find only a small energy gap between the "symmetric" doubly hydrogen-bonded and slightly displaced "staggered" structures, again in agreement with the crystal structure data. Further calculations of the total ab initio interaction energy show that this follows the electrostatic term in its orientational variation, this phenomenon of "electrostatic domination" being well known in hydrogen-bonded systems. The end on arginine nitrogen atoms are observed to be more surface-exposed than N epsilon, as demonstrated by their greater accessibilities over a large sample of proteins. This helps explain the side on and end on preferences of intra- and intermolecular interactions, respectively. We also note the effect of short sequence intervals, particularly i in equilibrium with i + 2 relationships, in forcing many intramolecular contacts to be side on.     

Refined structure of porcine pepsinogen at 1.8 A resolution

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.     

Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.     

Direct evidence for methyl group coordination by carbon-oxygen hydrogen bonds in the lysine methyltransferase SET7/9

SET domain lysine methyltransferases (KMTs) are S-adenosylmethionine (AdoMet)-dependent enzymes that catalyze the site-specific methylation of lysyl residues in histone and non-histone proteins. Based on crystallographic and cofactor binding studies, carbon-oxygen (CH · · · O) hydrogen bonds have been proposed to coordinate the methyl groups of AdoMet and methyllysine within the SET domain active site. However, the presence of these hydrogen bonds has only been inferred due to the uncertainty of hydrogen atom positions in x-ray crystal structures. To experimentally resolve the positions of the methyl hydrogen atoms, we used NMR (1)H chemical shift coupled with quantum mechanics calculations to examine the interactions of the AdoMet methyl group in the active site of the human KMT SET7/9. Our results indicated that at least two of the three hydrogens in the AdoMet methyl group engage in CH · · · O hydrogen bonding. These findings represent direct, quantitative evidence of CH · · · O hydrogen bond formation in the SET domain active site and suggest a role for these interactions in catalysis. Furthermore, thermodynamic analysis of AdoMet binding indicated that these interactions are important for cofactor binding across SET domain enzymes.     

Expression of DHA-Metabolizing Enzyme Alox15 is Regulated by Selective Histone Acetylation in Neuroblastoma Cells

The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is enriched in neural membranes of the CNS, and recent studies have shown a role of DHA metabolism by 15-lipoxygenase-1 (Alox15) in prefrontal cortex resolvin D1 formation, hippocampo-prefrontal cortical long-term-potentiation, spatial working memory, and anti-nociception/anxiety. In this study, we elucidated epigenetic regulation of Alox15 via histone modifications in neuron-like cells. Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate significantly increased Alox15 mRNA expression. Moreover, Alox15 expression was markedly upregulated by Class I HDAC inhibitors, MS-275 and depsipeptide. Co-treatment of undifferentiated SH-SY5Y cells with the p300 histone acetyltransferase (HAT) inhibitor C646 and TSA or sodium butyrate showed that p300 HAT inhibition modulated TSA or sodium butyrate-induced Alox15 upregulation. Differentiation of SH-SY5Y cells with retinoic acid resulted in increased neurite outgrowth and Alox15 mRNA expression, while co-treatment with the p300 HAT inhibitor C646 and retinoic acid modulated the increases, indicating a role of p300 HAT in differentiation-associated Alox15 upregulation. Increasing Alox15 expression was found in primary murine cortical neurons during development from 3 to 10 days-in-vitro, reaching high levels of expression by 10 days-in-vitro—when Alox15 was not further upregulated by HDAC inhibition. Together, results indicate regulation of Alox15 mRNA expression in neuroblastoma cells by histone modifications, and increasing Alox15 expression in differentiating neurons. It is possible that one of the environmental influences on the immature brain that can affect cognition and memory, may take the form of epigenetic effects on Alox15 and metabolites of DHA.     

Monensin A acid complexes as a model of electrogenic transport of sodium cation

New Monensin A acid complexes with water molecule, sodium chloride and sodium perchlorate were obtained and studied by X-ray and (1)H, (13)C NMR and FT-IR methods as well as ab initio calculations. The crystal structure of the complexes indicates the complexation of the water molecule and Na(+) cation in the pseudo-cycle conformation of the Monensin acid molecule stabilised by intramolecular hydrogen bonds. Important for stabilisation of this structure is also the intermolecular hydrogen bonds with water molecule or the coordination bonds with Na(+) cation. It is demonstrated that the counterions forming intermolecular hydrogen bonds with OH groups influence the strength of the intramolecular hydrogen bonds, but they have no influence on the formation of pseudo-cyclic structure. Spectroscopic studies of the complexes in dichloromethane solution have shown that the pseudo-cyclic structure of the compounds is conserved. As follows from the ab initio calculations, the interactions between the Na(+) cation and the electronegative oxygen atoms of Monensin acid totally change the molecular electrostatic potential around the supramolecular Monensin acid-Na(+) cationic complex relative to that of the neutral Monensin acid molecule.     

Acetyltransferase p300 inhibitor reverses hypertension‐induced cardiac fibrosis

Epigenetic dysregulation plays a crucial role in cardiovascular diseases. Previously, we reported that acetyltransferase p300 (ATp300) inhibitor L002 prevents hypertension‐induced cardiac hypertrophy and fibrosis in a murine model. In this short communication, we show that treatment of hypertensive mice with ATp300‐specific small molecule inhibitor L002 or C646 reverses hypertension‐induced left ventricular hypertrophy, cardiac fibrosis and diastolic dysfunction, without reducing elevated blood pressures. Biochemically, treatment with L002 and C646 also reverse hypertension‐induced histone acetylation and myofibroblast differentiation in murine ventricles. Our results confirm and extend the role of ATp300, a major epigenetic regulator, in the pathobiology of cardiac hypertrophy and fibrosis. Most importantly, we identify the efficacies of ATp300 inhibitors C646 and L002 in reversing hypertension‐induced cardiac hypertrophy and fibrosis, and discover new anti‐hypertrophic and anti‐fibrotic candidates.     

Quercetin suppresses cyclooxygenase-2 expression and angiogenesis through inactivation of P300 signaling

Quercetin, a polyphenolic bioflavonoid, possesses multiple pharmacological actions including anti-inflammatory and antitumor properties. However, the precise action mechanisms of quercetin remain unclear. Here, we reported the regulatory actions of quercetin on cyclooxygenase-2 (COX-2), an important mediator in inflammation and tumor promotion, and revealed the underlying mechanisms. Quercetin significantly suppressed COX-2 mRNA and protein expression and prostaglandin (PG) E(2) production, as well as COX-2 promoter activation in breast cancer cells. Quercetin also significantly inhibited COX-2-mediated angiogenesis in human endothelial cells in a dose-dependent manner. The in vitro streptavidin-agarose pulldown assay and in vivo chromatin immunoprecipitation assay showed that quercetin considerably inhibited the binding of the transactivators CREB2, C-Jun, C/EBPβ and NF-κB and blocked the recruitment of the coactivator p300 to COX-2 promoter. Moreover, quercetin effectively inhibited p300 histone acetyltransferase (HAT) activity, thereby attenuating the p300-mediated acetylation of NF-κB. Treatment of cells with p300 HAT inhibitor roscovitine was as effective as quercetin at inhibiting p300 HAT activity. Addition of quercetin to roscovitine-treated cells did not change the roscovitine-induced inhibition of p300 HAT activity. Conversely, gene delivery of constitutively active p300 significantly reversed the quercetin-mediated inhibition of endogenous HAT activity. These results indicate that quercetin suppresses COX-2 expression by inhibiting the p300 signaling and blocking the binding of multiple transactivators to COX-2 promoter. Our findings therefore reveal a novel mechanism of action of quercetin and suggest a potential use for quercetin in the treatment of COX-2-mediated diseases such as breast cancers.     

Structural investigation of the alpha-1-antichymotrypsin: prostate-specific antigen complex by comparative model building.

Prostate-specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate-derived member of which is human glandular kallikrein-1 (hK2). Active PSA and hK2 are both 237-residue kallikrein-like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and compared to PSA and six other serine proteases in order to analyze in depth the role of the surface-accessible loops surrounding the active site. The results show that PSA and hK2 share extensive structural similarity and that most amino acid replacements are centered on the loops surrounding the active site. Furthermore, the electrostatic potential surfaces are very similar for PSA and hK2. PSA interacts with at least two serine protease inhibitors (serpins): alpha-1-antichymotrypsin (ACT) and protein C inhibitor (PCI). Three-dimensional model structures of the uncleaved ACT molecule were developed based upon the recent X-ray structure of uncleaved antithrombin. The serpin was docked both to PSA and hK2. Amino acid replacements and electrostatic complementarities indicate that the overall orientation of the proteins in these complexes is reasonable. In order to investigate PSA's heparin interaction sites, electrostatic computations were carried out on PSA, hK2, protein C, ACT, and PCI. Two heparin binding sites are suggested on the PSA surface and could explain the enhanced complex formation between PSA and PCI, while inhibiting the formation of the ACT-PSA complex, PSA, hK2, and their preliminary complexes with ACT should facilitate the understanding and prediction of structural and functional properties for these important proteins also with respect to prostate diseases.     

Anticarcinogenic Bowman Birk inhibitor isolated from snail medic seeds (Medicago scutellata): solution structure and analysis of self-association behavior

The high-resolution three-dimensional structure of a Bowman Birk inhibitor, purified from snail medic seeds (Medicago scutellata) (MSTI), has been determined in solution by 1H NMR spectroscopy at pH 5.6 and 27 degrees C. The structure of MSTI comprises two distinct symmetric domains each composed of a three-stranded beta-sheet containing a VIb type loop, where the active sites are located. A characteristic geometry of three aromatic residues confers stability to this protein, and we observe that this feature is conserved in all the Bowman Birk inhibitors of known structure. The two active domains exhibit different conformational features: the second domain displays higher flexibility and hydrophobicity with respect to the first one, and these properties have been correlated to a lower trypsin inhibitory specificity, in agreement with titration studies that have shown a stoichiometric ratio MSTI:trypsin of 1:1.5. NMR analysis indicated that MSTI undergoes self-association at concentrations higher than 2 mM, and the residues involved in this mechanism are localized at opposite faces of the molecule, having the highest positive and negative potential, respectively, thus indicating that electrostatic intermolecular interactions are the driving forces for MSTI association. Most of the residues affected by self-association are highly conserved in BBIs from different seeds, suggesting a functional relevance for these charged superficial patches, possibly involved in the interaction with other enzymes or macromolecules, thus triggering anti-carcinogenic activity.     

Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

β-Lactamase-mediated resistance to β-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among β-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the β-lactamase family and probably for most carboxypeptidases.     

Substrate binding and catalytic mechanism in phospholipase C from Bacillus Cereus: A molecular mechanics and molecular dynamics study

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 Å) 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 AM1-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 AM1-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure. © 1997 John Wiley and Sons, Inc. Biopoly 42: 319–336, 1997