Backbone makes a significant contribution to the electrostatics of alpha/beta-barrel proteins.

The electrostatic properties of seven alpha/beta-barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose-1,6-bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the alpha/beta-barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of alpha/beta-barrel enzymes for negatively charged substrates.     

十二烷基硫酸钠对高压静电场对酶作用的影响

研究了十二烷基硫酸钠(SDS)对高压静电场对酶作用的影响。结果表明:在强度为3×103V/cm的电场作用下,十二烷基硫酸钠使过氧化氢酶对高压静电场的作用变得更加敏感。低浓度的十二烷基硫酸钠中,过氧化氢酶在高压静电场的作用下活力升高;0.04mmol/L～0.12mmol/L的十二烷基硫酸钠中,酶活力在高压静电场作用下先升高后下降;高于0.40mmol/L,则酶活力单调下降。十二烷基硫酸钠对高压静电场中的过氧化氢酶具有一定的保护作用。     

Surface charge measurements on Micrococcus lysodeikticus and the catalytic implications for lysozyme

Electrophoresis measurements on Micrococcus lysodeikticus have shown that the net surface charge density on the cell wall is constant at around -1.5 microC/cm2 for the pH range 4-8. This result has enabled a quantitative analysis to be made of how the electrostatic field associated with the negatively charged cell wall influences the ionic strength and pH dependency of the lytic activity of lysozyme towards M. lysodeikticus. A dominant effect is the creation of a local pH gradient at the cell wall, and at high ionic strengths the lytic activity is found to be controlled by an electrostatic force of attraction between the lysozyme molecule and the cell wall. As the ionic strength of the supporting electrolyte is decreased, however, an electrostatic force of repulsion becomes dominant and is associated with a negative charge carried by the lysozyme molecule, which could possibly be the ionized Asp-52 residue at the active site. This is considered to arise from the fact that at low ionic strengths the fine details of the heterogeneous charge distribution on the cell wall and lysozyme molecule are only partially screened by counter ions.     

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.     

Influence of external electrostatic fields on enzyme systems of phospholipid deacylation

The in vivo effect of external electrostatic field (200 kV/m for 1 h) on the activity of type A phospholipases and lysophospholipases of erythrocyte and mitochondrial membranes was studied. The electrostatic field exposure activated membrane-associated deacylation of phosphatidylcholine fraction in erythrocyte membranes.     

高压静电场对离子溶液中过氧化氢酶的作用研究

研究了 KBr、磷酸缓冲液、Na Cl对高压静电场对过氧化氢酶作用的影响。结果表明 :浓度在 0 .0 2 m mol/L～ 0 .0 5 m mol/L的低浓度的无机离子对高压静电场对过氧化氢酶的作用影响很小。浓度在 0 .1mmol/L～0 .4 0 m mol/L的无机离子对高压静电压对过氧化氢酶的活力影响较大 ,过氧化氢酶在 3× 10 3V /cm的电场作用下活力只会升高。而对照出现先下降后上升的现象。浓度高于 0 .80 m mol/L时 ,无机离子可以保护过氧化氢酶不受高压静电场的影响。无机离子的这种效应可能是无机离子在高压静电场的作用下 ,产生反抗电场降低了高压静电场的强度所致。     

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair

We present an analysis of the electrostatic properties in the catalytic site of papain (EC 3.4.22.2), an archetype enzyme of the C1 cysteine proteinase family, and we investigate their possible role in the formation, stabilization and regulation of the Cys25((-))...His159((+)) catalytic ion pair. The electrostatic properties were computed using a reassociation method based in multicentered multipolar expansions obtained from ab initio quantum calculations of overlapping protein fragments. Solvent effects were introduced by coupling the use of multicentered multipolar expansions to two continuum boundary element methods to solve the Poisson and the linearized Poisson-Boltzmann equations. The electrostatic profile found in the proton transfer region of papain showed that this enzyme has a well-defined electrostatic environment to favor the formation and stabilization of the catalytic ion pair. The papain catalytic site electrostatic profile can be considered as an electrostatic fingerprint of the papain family with the following characteristics: (i) the presence of a net electric field highly aligned in the (Cys25)-SG-->(His159)-ND1 direction; (ii) the electrostatic profile has a saddle-point character; (iii) it is basically a local environmental effect. Furthermore, our analysis describes a possible regulatory mechanism (the E(SG-->ND1) attenuation effect) controlling the ion pair reactivity and permits to infer the Asp57 acidic residue as the most probable candidate to act as the electrostatic modulator.     

Electrostatic fields near the active site of human aldose reductase: 1. New inhibitors and vibrational stark effect measurements

Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new hALR2 inhibitor was designed and synthesized that contains a nitrile probe with a Stark tuning rate of 0.77 cm-1/(MV/cm). Mutations to amino acid residues in the vicinity of the nitrile functional group were selected based on electrostatics calculations, possible complications from hydrogen bonds near the nitrile, and comparison with the active site of human aldehyde reductase, whose structure is very similar. Changes in the absorption energy of the nitrile probe when bound to those mutated proteins were then used to quantify perturbations to the protein's electrostatic field. Electrostatic field changes as large as -10 MV/cm were observed. Measured electrostatic fields were compared to predictions based on continuum electrostatics calculations, revealing that substantial modifications to the calculation strategy are necessary. The effects of hydrogen bonding of amino acid side chains to the nitrile probe are considered, and applications of vibrational Stark effect spectroscopy to investigations of ligand binding and biological function are discussed.     

高压静电场对过氧化氢酶的稳定作用及机理研究

用高压静电场直接处理过氧化氢酶溶液 ,结果表明 :不同强度的高压静电场能提高和降低过氧化氢酶的活力。电场强度越大 ,酶活力达到平衡所需时间越短。处理后的过氧化氢酶溶液 ,玻棒 30秒的搅拌作用能使酶活力基本上回到原来的状态。溶液极性降低 ,酶活力达到平衡所需时间减少。处于高压静电场中的过氧化氢酶溶液 ,在 2 0℃时 ,电场强度为 3× 10 3V/cm ,活力能保持 5～ 6天 ,而对照只能保持 2～ 3天。在 30℃时 ,同样电场强度下 ,活力能保持 48小时左右 ,对照能保持 2 4小时左右。经高压静电场处理的酶 ,活力保持时间也比对照长。研究表明 :高压静电场对酶活力的稳定作用很可能是高压静电场使酶及溶液发生极化作用产生的。     

pH Dependence of catalysis by Pseudomonas aeruginosa isochorismate-pyruvate lyase: implications for transition state stabilization and the role of lysine 42

An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H side chain at high pH, the enzyme retains lyase activity at ～100-fold lowered catalytic efficiency but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid side chains.     

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.     

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.     

Mechanism of reactant and product dissociation from the anthrax edema factor: a locally enhanced sampling and steered molecular dynamics study

The anthrax edema factor is a toxin overproducing damaging levels of cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) from ATP. Here, mechanisms of dissociation of ATP and products (cAMP, PPi) from the active site are studied using locally enhanced sampling (LES) and steered molecular dynamics simulations. Various substrate conformations and ionic binding modes found in crystallographic structures are considered. LES simulations show that PPi and cAMP dissociate through different solvent accessible channels, while ATP dissociation requires significant active site exposure to solvent. The ionic content of the active site directly affects the dissociation of ATP and products. Only one ion dissociates along with ATP in the two-Mg(2+) binding site, suggesting that the other ion binds EF prior to ATP association. Dissociation of reaction products cAMP and PPi is impaired by direct electrostatic interactions between products and Mg(2+) ions. This provides an explanation for the inhibitory effect of high Mg(2+) concentrations on EF enzymatic activity. Breaking of electrostatic interactions is dependent on a competitive binding of water molecules to the ions, and thus on the solvent accessibility of the active site. Consequently, product dissociation seems to be a two-step process. First, ligands are progressively solvated while preserving the most important electrostatic interactions, in a process that is dependent on the flexibility of the active site. Second, breakage of the electrostatic bonds follows, and ligands diffuse into solvent. In agreement with this mechanism, product protonation facilitates dissociation.     

Resistance of Dextran-Modified Hyaluronidase to Inhibition by Heparin

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.     

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.     

Electrostatics in the active site of an alpha-amylase.

The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for the Bacillus licheniformis alpha-amylase by constructing site-directed mutants that were predicted to change the pKa values of the catalytic residues and thus change the pH-activity profile of the enzyme. To change the pKa of the catalytic residues in the active site, we constructed mutations that altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule. The results show that changing the hydrogen bonding network near an active site residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity. The differences in the pH-activity profiles for these mutants were modest. pH-activity profiles of mutants which change the net charge on the molecule were significantly different from the wild-type pH-activity profile. The differences were, however, difficult to correlate with the electrostatic field changes calculated. In several cases we observed that pH-activity profiles shifted in the opposite direction compared to the shift predicted from electrostatic calculations. This strongly suggests that electrostatic effects cannot be solely responsible for the pH-activity profile of the B. licheniformis alpha-amylase.     

高压静电场对甜菜种子生物学效应作用机理的分析

选用甜菜种子为代表,通过实验室生理生化测定及现代物理仪器测定(以顺磁共振波谱技术测定自由基含量;以液闪仪单光子监测装置测定超弱发光强度)等方法来分析静电场对农作物种子生物效应的作用机理。实验结果表明:一定剂量的静电场能提高甜菜种子的发芽率、呼吸强度、自由基含量及超弱发光强度。通过分析实验结果,提出电场对农作物生物效应的作用机理:静电场是通过作用于种子体内的水分子来提高种子内的自由基含量而提高种子的超弱发光强度及生物膜的透性进而影响种子的生命活动的。     

Catalytic subunit of cAMP-dependent protein kinase: Electrostatic features and peptide recognition

The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5–24). The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit. Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine. These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M. J. Zoller and C. S. Gibbs [(1991) Journal of Biological Chemistry, Vol. 266, pp. 8923–8931; C. S. Gibbs and M. J. Zoller (1991) Biochemistry, Vol. 30, p. 22]. This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit. Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text. pK_a shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids. The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N. The calculated pK_a shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases. This prediction has been experimentally confirmed for the D166N mutant. The correlation between calculated electrostatic free energy changes and measured binding energy, and pK_a shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements. The calculations of electrostatic energy and ΔpK_a have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer. © 1996 John Wiley & Sons, Inc.     

Particle deposition onto a human head: Influence of electrostatic and wind fields

This study investigates electrostatic fields surrounding the human head and particle deposition onto facial skin and eyes caused by the combined effect of electrostatic and wind fields. The electrostatic fields are calculated by a three-dimensional numerical model calculating the field strength between a field source and a human head. The deposition velocity can be viewed as determined by the sum of two contributions: that of an electrostatic field and that of a wind field. Deposition velocities are calculated by a semiempirical particle deposition model that considers particle transport from the free stream to the human face. The particle deposition model uses the electrostatic field model results as input parameters and is applied to the forehead and eyes of two facial shapes for two different turbulence conditions and aerosol charge distributions. The results of different practical working conditions, under which the potential difference between head (person) and source ranges from 5.6 to 15.0 kV, indicates that the presence of electrostatic fields always increases particle deposition for industrial aerosols. For aged aerosols an effect is only present for submicron particles. Bioelectromagnetics 19:246–258, 1998. © 1998 Wiley-Liss, Inc.