Glycocalyx electrostatic potential profile analysis: ion, pH, steric, and charge effects

The Poisson-Boltzmann equation is modified to consider charge ionogenicity, steric exclusion, and charge distribution in order to describe the perimembranous electrostatic potential profile in a manner consistent with the known morphology and biochemical composition of the cell's glycocalyx. Exact numerical and approximate analytical solutions are given for various charge distributions and for an extended form of the Donnan potential model. The interrelated effects of ionic conditions, bulk pH, ion binding, local dielectric, steric volume exclusion, and charge distribution on the local potential, pH, and charge density within the glycocalyx are examined. Local charge-induced, potential-mediated pH reductions cause glycocalyx charge neutralization. Under certain conditions, local potentials may be insensitive to ionic strength or may decrease in spite of increasing charge density. The volume exclusion of the glycocalyx reduces the local ion concentration, thereby increasing the local potential. With neutral lipid membranes, the Donnan and surface potential agree if the glycocalyx charge distribution is both uniform and several times thicker than the Debye length (approximately 20 A in thickness under physiological conditions). Model limitations in terms of application to microdomains or protein endo- and ectodomains are discussed.     

Dielectric studies of the binding of water to lysozyme

Dielectric dispersion measurements as a function of hydration are reported for lysozyme powder. The dispersion that occurs in the frequency range 10 kHz to 10 GHz can be analysed in terms of bound water molecules that form single or multiple hydrogen bonds, and the numbers found in these two categories agree well with recent X-ray data for lysozyme crystals. The dielectric data also indicate that at 20% ($\text{w}\text{w}$) hydration the bound water acts as a plasticizer to increase the vibrational freedom of the protein structure, and that this may be of relevance to the fact that the onset of enzymatic activity occurs at this hydration level. Also, a sudden transition in the polarizability of the protein-water system is found to occur at 7% ($\text{w}\text{w}$) hydration.     

Theoretical studies of the electrostatic stability of A,B, C,and Z-DNA conformers as a function of monovalent ion concentration and ion type

Theoretical calculations using a two-state model of counterion phosphate interactions have been performed in order to determine the electrostatic stability of DNA in various ionic environments. Comparison of the stabilities of different conformations leads to results consistent with the B→Z transitions observed in high-salt environments and suggest that at very low ionic strnegths the left-handed Z(II) form is an electrostatically preferred conformation. An ion-type dependence of the high-salt B→Z transition midpoint is predicted. Calculations of the energies of the A and C conformations are also discussed in relation to experimentally observed transitions.     

Modeling the kinetics of enzymic reactions in mainly solid reaction mixtures

There is currently considerable interest in using mainly solid reaction mixtures for enzymic catalysis. In these reactions starting materials dissolve into, and product materials crystalize out of, a small amount of liquid phase in which the catalytic reaction occurs. An initial mathematical model for mass transfer effects in such systems is constructed using some physically reasonable approximations. The model equations are solved numerically to determine how the reactant concentrations vary with time and position. To evaluate the extent to which mass transfer limits the overall rate of product formation, an effectiveness factor is defined as the ratio of the observed total reaction rate to the total reaction rate in the reaction limited limit. As expected, the value of the effectiveness factor in steady state is strongly dependent on the Thiele modulus. However, it is also observed that the effectiveness factor can vary widely as a result of changes in the other dimensionless groups characterizing the system. For example, there are situations with Thiele modulus equal to unity in which the value of the effectiveness factor varies between approximately 0.1 and 0.8 as the other parameters are varied in physically reasonable ranges. Analytical asymptotic solutions that provide good approximations to the numerically calculated results in various physically important limiting cases are also presented.     

Electron-transfer reactions of photoreduced flavin analogues with c-type cytochromes: quantitation of steric and electrostatic factors

We have found correlations between rate constants and the difference in redox potential of the reactants for electron-transfer reactions between oxidized cytochromes and either photoproduced riboflavin or flavin mononucleotide (FMN) semiquinones (the latter rate constants extrapolated to infinite ionic strength). The riboflavin-cytochrome rate constants are about 70% of those for reduction by lumiflavin, probably because of steric interference by the ribityl side chain. Reduction of cytochromes by FMN semiquinone was ionic strength dependent in all cases, due to electrostatic interactions. Extrapolation of rate constants to infinite ionic strength shows that the phosphate exerts a significant steric effect as well (rate constants average about 27% of those for lumiflavin, although part of this decrease is due to a difference in the semiquinone pK value). Differences in the magnitude of the FMN steric effect correlate well with surface topology differences for those cytochromes whose three-dimensional structures are known. Mitochondrial cytochromes c and the cytochromes c2 all showed attractive (plus-minus) interaction with FMN in spite of the fact that some of these proteins have large net negative charges. Four small c-type cytochromes (including Pseudomonas cytochrome c-551) show a weak repulsive interaction with FMN semiquinone. We conclude that flavosemiquinones interact at a site on the cytochromes that is near the exposed heme edge. There is a large positive electrostatic field at this site in mitochondrial cytochrome c and the cytochromes c2, but this region is primarily hydrophobic in Pseudomonas cytochrome c-551 and in the other small bacterial cytochromes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A vector method of representation of enzymic reactions: 1. Parametric classification of types of enzymic reaction

A new “parametric” classification of the types of single-substrate enzymic reactions is proposed that includes 15 different types: seven inhibited, seven activated and one initial (uninhibited, i=0 and nonactivated, a=0). The new classification takes into account both the “parametricity” of these reactions and the mechanisms of their action. It unites all the types in an original symmetric system which vividly demonstrates the interconnection between separate (strictly definite) types of inhibited and activated enzymic reactions. The proposed classification permits the revision and some corrections to traditional “competitive” terminology as well as the application of these ideas and mathematical approaches pertinent to the calculation of reactions of enzyme inhibition for data analysis of enzyme activation.     

Xyloglucan-based diblock co-oligomer: Synthesis,self-assembly and steric stabilization of proteins

We propose a novel plant-based amphiphilic diblock co-oligomers (BCO) surfactant containing only carbohydrate segments and examine its potential as a biosourced stabilizer. The synthesis of an amphiphilic xyloglucan-based BCO, composed of a hydrophilic xyloglucan oligosaccharide (XGO) block “clicked” to a hydrophobic peracetylated XGO is described. Dynamic light scattering experiments correlated with transmission electron microscopy observations showed that this new class of amphiphilic BCO self-assembles in water to form spherical micelles with a hydrodynamic diameter of 22 nm. Preliminary studies indicate that the XGO-based BCO sterically stabilizes gliadin and zein nanoparticle suspensions. The stabilization results were compared to those using pluronic F-68, a commercial surfactant. For gliadin nanoparticles, both surfactants result in essentially the same morphology and polydispersity. However, for the zein nanoparticles, the XGO-based BCO stabilizer gave lower polydispersity.     

Theoretical studies of the electrostatic interactions in aspartic proteinases, intramolecular interactions in pepsin and penicillopepsin

A semi-empirical approach has been used to estimate the intramolecular electrostatic interactions in pepsin and penicillopepsin. The pH-dependence of the free energy electrostatic term was calculated, and the pH-dependence of the domain interactions has been estimated. As it was shown, the contribution of electrostatic interactions is rather small for the stabilization of the native structure. At the same time the electrostatic repulsion between domains increases with the increase of pH. The later can be the cause of the alkaline denaturation of pepsin and domain mobility.     

Catalysis in glycine N-methyltransferase: testing the electrostatic stabilization and compression hypothesis

Glycine N-methyltransferase (GNMT) is an S-adenosyl-l-methionine dependent enzyme that catalyzes glycine transformation to sarcosine. Here, we present a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study of the reaction compared to the counterpart process in water. The process takes place through an SN2 mechanism in both media with a transition state in which the transferring methyl group is placed in between the donor (SAM) and the acceptor (the amine group of glycine). Comparative analysis of structural, electrostatic, and electronic characteristics of the in-solution and enzymatic transition states allows us to get a deeper insight into the origins of the enzyme's catalytic power. We found that the enzyme is able to stabilize the substrate in its more active basic form by means of a positively charged residue (Arg175) placed in the active site. However, the maximum stabilization is attained for the transition state. In this case, the enzyme is able to form stronger hydrogen bonds with the positively charged amine group. Finally, we show that in agreement with previous computational studies on other methyltransferases, there is no computational evidence for the compression hypothesis, as was formulated by Schowen (Hegazi, M. F., Borchardt, R. T., and Schowen, R. L. (1979) J. Am. Chem. Soc. 101, 4359-4365).     

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.     

Differential electrostatic stabilization of A-, B-, and Z-forms of DNA

The static accessibility discrete charge algorithm for protein charge interactions is extended to the case of linear polyelectrolytes. In this model, the effective dielectric value between surface charge sites depends predominantly on the solvent ionic strength and the solvent accessibilities of the charge sites. This treatment accounts for the phenomena of specific ion binding in the context of a general electrostatic effect [Matthew and Richards (1982) Biochemistry 21 , 4989]. Specific ion sites are determined by locating areas of high electrostatic potential at the solvent interface of the macromolecule. At a given ionic strength the calculated potential at a site is taken to describe a binding constant and therefore the ion site occupancy. For a 20-base-pair fragment of B-DNA, net charge of ?40, 16 ion sites are indicated in the minor groove. The partial occupancy of each site increases from 0.2 to 0.5 as the ionic strength is increased from 0.01 to 0.50. Over the same range of ionic strength, the electrostatic free energy of this charge array is calculated to change from +0.6 to ?0.05 kcal/bp. Parallel behavior is predicted for A- and Z-DNA charge geometries. The most stable configuration, based on electrostatic criteria, at high ionic strength (I = 0.1–0.5) is that of Z-DNA. In this range, the ratio of “bound” sodium to phosphate is predicted to be less than 0.4.     

Theoretical study of the distal-side steric and electrostatic effects on the vibrational characteristics of the FeCO unit of the carbonylheme proteins and their models.

The vibronic theory of activation and quantum chemical intermediate neglect of differential overlap (INDO) calculations are used to study the activation of carbon monoxide (change of the C-O bond index and force field constant) by the imidazole complex with heme in dependence on the distortion of the porphyrin ring, geometry of the CO coordination, iron-carbon and iron-imidazole distances, iron displacement out of the porphyrin plane, and presence of the charged groups in the heme environment. It is shown that the main contribution to the CO activation stems from the change in the sigma donation from the 5 sigma CO orbital to iron, and back-bonding from the iron to the 2 pi orbital of CO. It follows from the results that none of the studied distortions can explain, by itself, the wide variation of the C-O vibrational frequency in the experimentally studied model compounds and heme proteins. To study the dependence of the properties of the FeCO unit on the presence of charged groups in the heme environment, the latter are simulated by the homogeneous electric field and point charges of different magnitude and location. The results show that charged groups can strongly affect the strength of the C-O bond and its vibrational frequency. It is found that the charges located on the distal side of the heme plane can affect the Fe-C and C-O bond indexes (and, consequently, the Fe-C and C-O vibrational frequencies), both in the same and in opposite directions, depending on their position. The theoretical results allow us to understand the peculiarities of the effect of charged groups on the properties of the FeCO unit both in heme proteins and in their model compounds.     

Mechanism and stereoelectronic effects in the lysozyme reaction

Lysozyme occupies a special place in the history of enzymology as the first enzyme to have its three-dimensional crystal structure elucidated by Phillips and co-workers in 1965. The crystallography, and much biochemical work, revealed three factors likely to be important for the mechanism of action: catalysis by the carboxyl group of Glu-35, catalysis by the ionized carboxyl group of Asp-52, and the conformation of the bound polysaccharide substrate. The work of the last 20 years has defined likely roles for the catalytic groups, but discussion of the conformational question came to a head only very recently with the suggestion that the fundamental stereoelectronic requirements of the glycoside-cleavage reaction might be decisive. Recent work on all three interlinked factors are reviewed.     

Theoretical studies of the electrostatic potential of some enzyme inhibitors using computer graphics techniques

A molecular graphics program is described for the depiction of electrostatic potentials on the van der Waals surface of molecules, using colored polygons. An example is given on the application of this method to the study of coumarins and flavones that inhibit the enzyme Glyoxalase I.     

Rapid reaction studies on the oxygenation reactions of catechol dioxygenase

The reaction of oxygen with catechol 1,2-dioxygenase from Pseudomonas arvilla ATCC 23974 in complex with catechol, 4-methylcatechol, and 4-fluorocatechol has been studied using single turnover stopped flow spectrophotometry. Two sequential enzyme intermediates have been resolved and their visible spectra characterized by computer-assisted methods. These intermediates are spectrally similar to those observed in a similar study with protocatechuate dioxygenase (Bull, C., Ballou, D. P., and Otsuka, S. J. Biol. Chem. 256, 12681-12686 (1981), although the first intermediate seen with the latter enzyme was not observed in this study. The rate of formation of intermediate I is oxygen-dependent and also accelerated by electron-donating substituents on the C-4 of the substrate. This is consistent with the proposed substrate reduction of dioxygen to form a hydroperoxide. Intermediate I is thus suggested to be a 6-hydroperoxycyclohexa-3,5-diene-1-one. The decay of intermediate I is also accelerated by electron donors and is consistent with the rearrangement of intermediate hydroperoxide via an acyl migration mechanism. It is inconsistent with mechanisms involving nucleophilic attack at the carbonyl carbon. Intermediate II is proposed to be an enzyme-product complex based on the resemblance of its visible spectra to those of the benzoate complex of catechol 1,2-dioxygenase and enzyme-product complexes of protocatechuate dioxygenase. Careful 18O2-labeling experiments have shown that no label is lost to the solvent, implying that no free hydroxide forms during catalysis.