Cofactor Ca2 Research Articles

Oligo-Adenine and Cyanuric Acid Supramolecular DNA-Based Hydrogels Exhibiting Acid-Resistance and Physiological pH-Responsiveness.

Expanding the functions and applications of DNA by integrating noncanonical bases and structures into biopolymers is a continuous scientific effort. An adenine-rich strand (A-strand) is introduced as functional scaffold revealing, in the presence of the low-molecular-weight cofactor cyanuric acid (CA, pKa 6.9), supramolecular hydrogel-forming efficacies demonstrating multiple pH-responsiveness. At pH 1.2, the A-strand transforms into a parallel A-motif duplex hydrogel cross-linked by AH+-H+A units due to the protonation of adenine (pKa 3.5). At pH 5.2, and in the presence of coadded CA, a helicene-like configuration is formed between adenine and protonated CA, generating a parallel A-CA triplex cross-linked hydrogel. At pH 8.0, the hydrogel undergoes transition into a liquid state by deprotonation of CA cofactor units and disassembly of A-CA triplex into its constituent components. Density functional theory calculations and molecular dynamics simulations, supporting the structural reconfigurations of A-strand in the presence of CA, are performed. The sequential pH-stimulated hydrogel states are rheometrically characterized. The hydrogel framework is loaded with fluorescein-labeled insulin, and the pH-stimulated release of insulin from the hydrogel across the pH barriers present in the gastrointestinal tract is demonstrated. The results provide principles for future application of the hydrogel for oral insulin administration for diabetes.

An autocatalytic multicomponent DNAzyme nanomachine for tumor-specific photothermal therapy sensitization in pancreatic cancer

Multicomponent deoxyribozymes (MNAzymes) have great potential in gene therapy, but their ability to recognize disease tissue and further achieve synergistic gene regulation has rarely been studied. Herein, Arginylglycylaspartic acid (RGD)-modified Distearyl acylphosphatidyl ethanolamine (DSPE)-polyethylene glycol (PEG) (DSPE-PEG-RGD) micelle is prepared with a DSPE hydrophobic core to load the photothermal therapy (PTT) dye IR780 and the calcium efflux pump inhibitor curcumin. Then, the MNAzyme is distributed into the hydrophilic PEG layer and sealed with calcium phosphate through biomineralization. Moreover, RGD is attached to the outer tail of PEG for tumor targeting. The constructed nanomachine can release MNAzyme and the cofactor Ca2+ under acidic conditions and self-assemble into an active mode to cleave heat shock protein (HSP) mRNA by consuming the oncogene miRNA-21. Silencing miRNA-21 enhances the expression of the tumor suppressor gene PTEN, leading to PTT sensitization. Meanwhile, curcumin maintains high intracellular Ca2+ to further suppress HSP-chaperone ATP by disrupting mitochondrial Ca2+ homeostasis. Therefore, pancreatic cancer is triple-sensitized to IR780-mediated PTT. The in vitro and in vivo results show that the MNAzyme-based nanomachine can strongly regulate HSP and PTEN expression and lead to significant pancreatic tumor inhibition under laser irradiation.

Cryo-EM structure of the prothrombin-prothrombinase complex

AbstractThe intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa–fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.

Synthetic Inhibitors of Snake Venom Enzymes: Thioesters Derived from 2-Sulfenyl Ethylacetate.

Snakebite envenomings are a global public health issue. The therapy based on the administration of animal-derived antivenoms has limited efficacy against the venom-induced local tissue damage, which often leads to permanent disability. Therefore, there is a need to find inhibitors against toxins responsible for local damage. This work aimed to synthesize thioesters derived from 2-sulfenyl ethylacetate and to evaluate the inhibitory effects on two snake venom toxins. Ethyl 2-((4-chlorobenzoyl)thio)acetate (I), Ethyl 2-((3-nitrobenzoyl)thio)acetate (II) and Ethyl 2-((4-nitrobenzoyl)thio)acetate (III) were synthesized and spectroscopically characterized. Computational calculations were performed to support the study. The inhibitory capacity of compounds (I–III) was evaluated on a phospholipase A2 (Cdcum6) isolated from the venom of the Colombian rattlesnake Crotalus durissus cumanensis and the P-I type metalloproteinase Batx-I isolated from Bothrops atrox. I–III inhibited PLA2 with IC50 values of 193.2, 305.4 and 132.7 µM, respectively. Otherwise, compounds II and III inhibited the proteolytic activity of Batx-I with IC50 of 2774 and 1879 µM. Molecular docking studies show that inhibition of PLA2 may be due to interactions of the studied compounds with amino acids in the catalytic site and the cofactor Ca2+. Probably, a blockage of the hydrophobic channel and some amino acids of the interfacial binding surface of PLA2 may occur.

Seleksi dan Optimasi Karakter Fisik Bakteri Penghasil Fitase yang Diisolasi dari Sumber Air Panas di Guci, Tegal

<p>The aimed of this study to isolate, select and characterize physically phytase-producing bacteria<br />from hot springs in the Guci, Tegal. Isolation and enrichment of bacteria using Lurya Betani media<br />with 2% of phytic acid substrate. Extraction of extracellular phytase thus obtained extract coarse and<br />optimization by loking at the relative activity at the difference in the resulting product. Phytaseproducing<br />bacteria can be isolated from the hot springs of the Guci, Tegal with the highest relative<br />activity in AG2 and AG2-1 colonies. The highest relative activity at a temperature of 55°C, pH 6, the<br />incubation time of 90 minutes, the substrate concentration of 3% and a metal cofactor Ca2+ at a<br />concentration of 10-4M. Phytase-producing bacteria can be isolated from a hot spring in Guci, Tegal<br />and have certain physical characteristics.</p>

Intradomain Allosteric Network Modulates Calcium Affinity of the C-Type Lectin Receptor Langerin.

Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca(2+) controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca(2+) affinity depending on the pH, thereby promoting fast ligand release.

Structural insight into signal conversion and inactivation by NTPDase2 in purinergic signaling

Cell surface-located nucleoside triphosphate diphosphohydrolases (NTPDase1, -2, -3, and -8) are oligomeric integral membrane proteins responsible for signal conversion and inactivation in extracellular nucleotide-mediated "purinergic" signaling. They catalyze the sequential hydrolysis of the signaling molecule ATP via ADP to AMP. Here we present the structure of the extracellular domain of Rattus norvegicus NTPDase2 in an active state at resolutions between 1.7 A and 2.1 A in four different forms: (i) apo form, (ii) ternary complex with the nonhydrolyzable ATP analog AMPPNP and cofactor Ca(2+), (iii) quaternary complex with Ca(2+) and bound products AMP and phosphate, and (iv) binary product complex with AMP only. Analysis of the ATP (analog) binding mode explains the importance of several residues for activity and allows suggestion of a catalytic mechanism. The carboxylate group of E165 serves as a catalytic base and activates a water molecule, which is well positioned for nucleophilic attack on the terminal phosphate. Based on analysis of the two product complex structures in which AMP adopts different conformations, a substrate binding mode for ADP hydrolysis is proposed. This allows for an understanding of how the same hydrolytic site can be engaged in ATP and ADP but not AMP hydrolysis.

Structural changes in the Mn4Ca cluster and the mechanism of photosynthetic water splitting

Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn4Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn4Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn-Mn and Ca(Sr)-Mn distances in the four intermediate S states, S0 through S3, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn4Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn-Mn and Ca(Sr)-Mn distances, on the S2-to-S3 and S3-to-S0 transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn-Mn and Mn-Ca(Sr) atoms in the O-O bond formation. Because PS II cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor.

Identification of Vascular Endothelial Growth Factor Receptor-binding Protein in the Venom of Eastern Cottonmouth: A NEW ROLE OF SNAKE VENOM MYOTOXIC LYS49-PHOSPHOLIPASE A2

Vascular endothelial growth factor (VEGF165) and its receptor KDR (kinase insert domain-containing receptor) are central regulators of blood vessel formation. We herein report a KDR-binding protein we have isolated in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49-phosholipase A2 (Lys49PLA2), an inactive PLA2 homologue with strong myotoxicity, in which Lys49 substitutes Asp49, a key residue for binding the essential cofactor Ca2+. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. KDR-bp also binds to a lesser extent with Flt-1 and IgG but not to other receptors with similar immunoglobulin-like domain structures such as platelet-derived growth factor receptor alpha. The interaction between KDR-bp and KDR was blocked by VEGF165, and KDR-bp specifically inhibited VEGF165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. Lys49PLA2s from another snake venom were found to exhibit similar receptor binding properties to KDR-bp. This is the first report to demonstrate that an exogenous factor antagonizes VEGF and its receptor system. Our observation offers further insight into the as yet unknown molecular mechanism of myotoxic activity of snake venom Lys49PLA2s. Furthermore, KDR-bp would make a valuable tool for studying the structure and function of KDR, such as that expressed on skeletal muscle cells.

Ca cofactor of the water-oxidation complex: Evidence for a Mn/Ca heteronuclear cluster

Calcium and chloride are necessary cofactors for the proper function of the oxygen-evolving complex (OEC) of Photosystem II (PS II). Located in the thylakoid membranes of green plants, cyanobacteria and algae, PS II and the OEC catalyze the light-driven oxidation of water into dioxygen (released into the biosphere), protons and electrons for carbon fixation. The actual chemistry of water oxidation is performed by a cluster of four manganese atoms, along with the requisite cofactors Ca{sup 2+} and Cl{sup -}. While the Mn complex has been extensively studied by X-ray absorption techniques, comparatively less is known about the Ca{sup 2+} cofactor. The fewer number of studies on the Ca{sup 2+} cofactor have sometimes relied on substituting the native cofactor with strontium or other metals, and have stirred some debate about the structure of the binding site. past efforts using Mn EXAFS on Sr-substituted PSII are suggestive of a close link between the Mn cluster and Sr, within 3.5 {angstrom}. The most recent published study using Sr EXAFS on similar samples confirms this finding of a 3.5 {angstrom} distance between Mn and Sr. This finding was base3d on a second Fourier peak (R {approx} 3 {angstrom}) in the Sr EXAFS from functionalmore » samples, but is absent from inactive, hydroxylamine-treated PS II. This Fourier peak II was found to fit best to two Mn at 3.5 {angstrom} rather than lighter atoms (carbon). Nevertheless, other experiments have given contrary results. They wanted to extend the technique by using polarized Sr EXAFS on layered Sr-substituted samples, to provide important angle information. Polarized EXAFS involves collecting spectra for different incident angles ({theta}) between the membrane normal of the layered sample and the X-ray electric field vector. Dichroism in the EXAFS can occur, depending on how the particular absorber-backscatterer (A-B) vector is aligned with the electric field. Through analysis of the dichroism, they extract the average number of scatterers per absorbing atom (N{sub iso}). Constraints on the structural model are then imposed by these parameters. In a complementary and definitive experiment, they use Ca K-edge EXAFS studies to probe the binding site of the native cofactor for any nearby Mn, within {approx} 4 {angstrom}. This is analogous to the Sr EXAFS studies already published, but it focuses on the native cofactor and avoids the treatments involving Ca depletion and Sr substitution. The samples examined were PS II membrane particles from spinach. This new technique promises to be a more sensitive and direct probe of the calcium binding site in PS II than Sr EXAFS. Clarifying whether the Ca cofactor is proximate to the Mn cluster, and finding its coordination environment at the various intermediate S-states of the OEC will reveal its important role in oxygen evolution.« less

Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

Manganese Oxyl Radical Intermediates and O−O Bond Formation in Photosynthetic Oxygen Evolution and a Proposed Role for the Calcium Cofactor in Photosystem II

Spin state considerations are proposed to sharply limit the possible O−O bond-forming steps in water oxidation by the oxygen evolving center of Photosystem II. A series of intermediates are proposed for the Kok S states on the basis of quantum chemical studies on simple model complexes; these are also consistent with the main biophysical data. Only one Mn atom in the active site cluster is thought to be redox-active and mediate O−O bond formation. A key concept is the formation of an unreactive MnO oxo at the S2 state, followed by its conversion to a reactive Mn−O• oxyl form at the S3 level, with radical character on the oxyl oxygen, at which point O−O bond formation can occur by a coupling between the oxyl and an outer-sphere water molecule. An MnOOH intermediate at S3 is proposed to lose a hydrogen atom to give O2. The role of the Ca cofactor is to bring about a 5- to 6-coordination change at S2, necessary for formation of a reactive oxo in S3. The chloride cofactor is assigned the role of charge neutralization.

Role of the cofactor calcium in the activation of outer membrane phospholipase A.

The enzymatic activity of the outer membrane phospholipase A (OMPLA), an integral membrane protein of Escherichia coli, is regulated by dimerization for which the cofactor Ca2+ is required. In this study, the interaction of Ca2+ with OMPLA was characterized, with an emphasis on the role of the cofactor in the activation process and dimerization. Kinetic experiments were done in which the enzyme was solubilized in mixed micelles of substrate and different detergents. It appeared that the affinity of OMPLA for Ca2+ was high (12 microM) if alkylphosphocholines were used as detergent, moderate (62 microM) if sulfobetaines were used, and very low (24 mM) if alkylpolyoxyethylene glycols were used. These results show that there is a strong modulation of the calcium binding properties of OMPLA by the lipid environment. In the presence of hexadecylphosphocholine micelles, the affinity of OMPLA for Ca2+ was determined by three direct binding techniques. Using gel filtration, it appeared that OMPLA has one high-affinity site (Kd approximately 36 microM) and a second site with moderate affinity (Kd approximately 358 microM). Sulfonylated-OMPLA, in which the active site serine had been covalently modified with hexadecanesulfonylfluoride, was used as a mimic for the acyl-enzyme intermediate. In gel filtration experiments, this sulfonylated-OMPLA displayed binding of two Ca2+ per enzyme monomer both with similar high affinity (Kd approximately 48 microM), indicative of a strong synergistic effect of active site occupation and the affinity of the second Ca2+ binding site. Isothermal titration calorimetric measurements confirmed only the presence of a high-affinity Ca2+ binding site, whereas in fluorescence experiments only the binding of the second Ca2+ could be observed. Chemical cross-linking was applied to investigate which of the two Ca2+ sites is involved in dimerization. OMPLA was monomeric in the absence of Ca2+, whereas already at low Ca2+ concentrations the enzyme was converted to its dimeric form. Therefore, we suggest that the first Ca2+ plays a role in the stabilization of the dimeric state of the enzyme. The role of the second Ca2+ and the observed synergy between active site occupancy and Ca2+ affinity are discussed.

A SequenceSpace analysis of Lys49 phopholipases A2: clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity.

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a sub-family of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Dimerization Regulates the Enzymatic Activity of Escherichia coli Outer Membrane Phospholipase A

The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca2+ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.

Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein.

Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.

Interaction of native and modified Naja melanoleuca phospholipases A2 with the fluorescent probe 8-anilinonaphtalene-1-sulfonate

The fluorescent probe 8-anilinonaphtalene-1-sulfonate (ANS) binds at the active site of the Naja melanoleuca snake venom phospholipase A2, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100 microM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2+. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pKa value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein. Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N. melanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.

Chemical modification of Lys-6 in phospholipase A2 from Naja melanoleuca snake venom.

Phospholipase A2 isolated from Naja melanoleuca snake venom was modified with 4-chloro-3,5-dinitrobenzoate. The modification reaction was accelerated by the cofactor Ca2+. Micellar concentrations of the substrate analog n-tetradecylphosphocholine retarded the inactivation of the enzyme. Modification resulted in the incorporation of one mole dinitrobenzoate/mole of protein. The modified residue could be identified as Lys-6 in the amino acid sequence of the enzyme. Although the modified enzyme still retains a high affinity for micellar substrate analogs and the cofactor Ca2+, it had lost nearly all its activity towards micellar substrates. Upon reduction of the nitro groups in the modified protein the enzymatic activity could be restored by more than 50%. Therefore Lys-6 cannot be directly involved in enzymatic catalysis. It is concluded that introduction of the negatively charged dinitrobenzoate group on Lys-6 gives rise to a distortion of the active site of the enzyme.

Binding of porcine pancreatic phospholipase A2 to various micellar substrate analogues. Involvement of histidine-48 and aspartic acid-49 in the binding process.

The interaction of porcine pancreatic phospholipase A2 (PA2) with micelles of various single-chain phospholipid analogues was studied by ultraviolet absorption difference spectroscopy and light-scattering measurements. The phospholipids used were either substrate analogues or products, varying in hydrocarbon chain lengths and polar head groups. The results indicate that the enzyme forms a stable complex over a wide range of enzyme and lipid concentrations. From the equivalent "molecular weight" and from the lipid to enzyme molar ratio (N) of the micelle--enzyme complex, it can be calculated that complexes containing saturated hydrocarbon chain lipids generally consist of two enzyme molecules and half of the number of lipid monomers present in free micelles. The interaction forces between the enzyme and lipid monomers bound in the complex are mainly hydrophobic. Stronger binding is found when the essential cofactor Ca2+ is bound to the enzyme. pH-titration studies on the binding of native PA2 to aggregated lipid structures showed that at least one group with a pKA value of 6.25 is involved in the interaction with lipid micelles. At acidic pH, micelle binding is stronger than at neutral or alkaline pH. Alkylation of the active site residue His48 resulted in a shift of the pKA value to 4.6, while addition of Ca2+ appears to stabilize the micelle-binding conformation of both native and modified enzymes over a broad pH range (pH 4--9.5). From these observations it is suggested that both the Ca2+ binding residue Asp49 [Fleer, E. A. M., Verheij, H. M., & de Haas, G. H. (1980) Eur. J. Biochem. 113, 283--288] and His48 control micelle binding of the native enzyme. For optimal binding in the absence of Ca2+, a long-distance hydrogen bond between these two residues is required; this can be established via a water molecule. It is assumed that it is a proton of this "H bond" which is titrated with a pKA value of 6.25. When the "H bond" is absent, as in the alkylated enzymes, Asp49 alone controls micelle binding with a pKA of 4.6. These results, together with the effect of Ca2+ on micelle binding, indicate that it is not the "hydrogen bridge" between His48 and Asp49 which is of main importance for an optimum binding conformation of the enzyme but the effective charge in the microenvironment of Asp49. It is proposed that a negative charge on this carboxylate causes a conformational change of the enzyme which leads to a protein conformation lacking an active micelle binding site. Binding of Ca2+ or reprotonation neutralizes this negative charge and restores the enzyme's ability to bind micelles.

Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.

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.