Electrostatic fields near the active site of human aldose reductase: 2. New inhibitors and complications caused by hydrogen bonds

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 nitrile-containing inhibitor was designed and synthesized, and the X-ray structure of its complex, along with cofactor NADP(+), with wild-type hALR2 was determined at 1.3 ? resolution. The nitrile is found to be in the proximity of T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to wild-type hALR2, indicating that the nitrile probe experiences two different microenvironments, and these could be empirically separated into a hydrogen-bonded and non-hydrogen-bonded population by comparison with the T113A mutant, in which a hydrogen bond to the nitrile is not present. Classical molecular dynamics simulations based on the structure predict a double-peak distribution in protein electric fields projected along the nitrile probe. The interpretation of these two peaks as a hydrogen bond formation-dissociation process between the probe nitrile group and a nearby amino acid side chain is used to explain the observation of two IR bands, and the simulations were used to investigate the molecular details of this conformational change. Hydrogen bonding complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect, and the consequences of this complication are discussed.     

Electrostatic effects of mutations of Ras glutamine 61 measured using vibrational spectroscopy of a thiocyanate probe

Mutations of human oncoprotein p21(Ras) (hereafter Ras) at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that electrostatically stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. We have tested the interactions between amino acids at this position and water by measuring changes in the electrostatic field experienced by a nitrile probe positioned near Ras Q61 using vibrational Stark effect (VSE) spectroscopy. We mutated this glutamine to every amino acid except cysteine and proline and then incubated these mutants with a Ral guanine nucleotide dissociation stimulator (Ral) containing the I18C mutation that was chemically labeled with a thiocyanate vibrational spectroscopic probe. The formation of the docked Ras Q61X-labeled Ral complex was confirmed by measurement of the dissociation constant of the interaction. We measured the absorption energy of this nitrile to determine any differences in electrostatic environment in the immediate vicinity of the thiocyanate probe between wild type and mutants of Ras. For each Ras Q61X mutant, we correlate the change in electrostatic field at position 61 with the solvent accessible surface area of polar components of the mutant side chain determined from a Boltzmann-weighted ensemble of structures, as well as the residue's hydration potential. These results support the hypothesis that the role of Ras Q61 is to stabilize water in or near the active site during GTP hydrolysis. The substantial effect that nonpolar side chains of Ras Q61X have on the absorption energy of the thiocyanate must be investigated with further experiments.     

Factors determining electrostatic fields in molecular dynamics simulations of the Ras/effector interface

Using molecular dynamics simulations, we explore geometric and physical factors contributing to calculated electrostatic fields at the binding surface of the GTPase Ras with a spectroscopically labeled variant of a downstream effector, the Ras-binding domain of Ral guanine nucleotide dissociation stimulator (RalGDS). A related system (differing by mutation of one amino acid) has been studied in our group using vibrational Stark effect spectroscopy, a technique sensitive to electrostatic fields. Electrostatic fields were computed using the AMBER 2003 force field and averaged over snapshots from molecular dynamics simulation. We investigate geometric factors by exploring how the orientation of the spectroscopic probe changes on Ras-effector binding. In addition, we explore the physical origin of electrostatic fields at our spectroscopic probe by comparing contributions to the field from discrete components of the system, such as explicit solvent, residues on the Ras surface, and residues on the RalGDS surface. These models support our experimental hypothesis that vibrational Stark shifts are caused by Ras binding to its effector and not the structural rearrangements of the effector surface or probe reorientation on Ras-effector binding, for at least some of our experimental probes. These calculations provide physical insight into the origin, magnitude, and importance of electrostatic fields in protein-protein interactions and suggest new experiments to probe the field's role in protein docking.     

Density functional and electrostatics study of oxidized and reduced ribonucleotide reductase; comparisons with methane monooxygenase

A combined broken symmetry density functional and electrostatics approach has been used to examine the active sites of the resting (RNR(ox)) and reduced (RNR(red)) forms of class I type ribonucleotide reductase in the protein and solvent environment. Active site cluster geometries and Heisenberg J values are discussed in the context of the available protein data. The total electrostatic interaction energy in the protein comprises a large reaction field component and a much smaller protein field term, the former suggesting strong dielectric polarization between the cluster and protein-solvent dielectrics; the latter is indicative of a very weak link to the protein environment. Decomposition of the protein field term elucidates the major electrostatic interactions between amino acid residues in the RNR R2 local environment and the active site cluster, enabling an energetic comparison of structurally equivalent residues with a related diiron protein, methane monooxygenase.     

Vibrational Stark effects calibrate the sensitivity of vibrational probes for electric fields in proteins

Infrared spectroscopy is widely used to probe local environments and dynamics in proteins. The introduction of a unique vibration at a specific site of a protein or more complex assembly offers many advantages over observing the spectra of an unmodified protein. We have previously shown that infrared frequency shifts in proteins can arise from differences in the local electric field at the probe vibration. Thus, vibrational frequencies can be used to map electric fields in proteins at many sites or to measure the change in electric field due to a perturbation. The Stark tuning rate gives the sensitivity of a vibrational frequency to an electric field, and for it to be useful, the Stark tuning rate should be as large as possible. Vibrational Stark effect spectroscopy provides a direct measurement of the Stark tuning rate and allows a quantitative interpretation of frequency shifts. We present vibrational Stark spectra of several bond types, extending our work on nitriles and carbonyls and characterizing four additional bond types (carbon-fluorine, carbon-deuterium, azide, and nitro bonds) that are potential probes for electric fields in proteins. The measured Stark tuning rates, peak positions, and extinction coefficients provide the primary information needed to design amino acid analogues or labels to act as probes of local environments in proteins.     

Catalytic efficiency of dehaloperoxidase A is controlled by electrostatics – application of the vibrational Stark effect to understand enzyme kinetics

The vibrational Stark effect is gaining popularity as a method for probing electric fields in proteins. In this work, we employ it to explain the effect of single charge mutations in dehaloperoxidase-hemoglobin A (DHP A) on the kinetics of the enzyme. In a previous communication published in this journal (BBRC 2012, 420, 733–737) it has been shown that an increase in the overall negative charge of DHP A through mutation causes a decrease in its catalytic efficiency. Here, by labeling the protein with 4-mercaptobenzonitrile (MBN), a Stark probe molecule, we provide further evidence that the diffusion control of the catalytic process arises from the electrostatic repulsion between the enzyme and the negatively charged substrate. The linear correlation observed between the nitrile stretching frequency of the protein-bound MBN and the catalytic efficiency of the single-site mutants of the enzyme indicates that electrostatic interactions play a dominant role in determining the catalytic efficiency of DHP A.     

Sampling field heterogeneity at the heme of c-type cytochromes by spectral hole burning spectroscopy and electrostatic calculations

We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations. The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes. In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed. The yeast species, on the other hand, displays a single electronic origin. Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space. The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes.     

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.     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.     

Conservation of electrostatic properties within enzyme families and superfamilies

Electrostatic interactions play a key role in enzyme catalytic function. At long range, electrostatics steer the incoming ligand/substrate to the active site, and at short distances, electrostatics provide the specific local interactions for catalysis. In cases in which electrostatics determine enzyme function, orthologs should share the electrostatic properties to maintain function. Often, electrostatic potential maps are employed to depict how conserved surface electrostatics preserve function. We expand on previous efforts to explain conservation of function, using novel electrostatic sequence and structure analyses of four enzyme families and one enzyme superfamily. We show that the spatial charge distribution is conserved within each family and superfamily. Conversely, phylogenetic analysis of key electrostatic residues provide the evolutionary origins of functionality.     

The relationship between physical property and function of highly activated mutants of thermolysin

Thermolysin mutants having a variety of amino acid at the 119th position are designed by considering electrostatic field effect upon the active area. The most activated mutant has five times higher hydrolytic activity than the wild type. Negative correlation between the activity and the thermal stability is observed. A combined effect of the flexibility of the substrate binding site and the negative electrostatic field around the site is suggested as a key to enhance the activity.     

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.     

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.     

Electrostatic recognition in substrate binding to serine proteases

Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.     

Probing electric fields in protein cavities by using the vibrational stark effect of carbon monoxide

To determine the magnitude and direction of the internal electric field in the Xe4 cavity of myoglobin mutant L29W-S108L, we have studied the vibrational Stark effect of carbon monoxide (CO) using infrared spectroscopy at cryogenic temperatures. CO was photodissociated from the heme iron and deposited selectively in Xe4. Its infrared spectrum exhibits Stark splitting into two bands associated with CO in opposite orientations. Two different photoproduct states can be distinguished, C' and C', with markedly different properties. For C', characteristic temperature-dependent changes of the area, shift, and width were analyzed, based on a dynamic model in which the CO performs fast librations within a double-well model potential. For the barrier between the wells, a height of approximately 1.8 kJ/mol was obtained, in which the CO performs oscillations at an angular frequency of approximately 25 cm(-1). The magnitude of the electric field in the C' conformation was determined as 11.1 MV/cm; it is tilted by an angle of 29 degrees to the symmetry axis of the potential. Above 140 K, a protein relaxation leads to a significantly altered photoproduct, C', with a smaller Stark splitting and a more confining potential (barrier >4 kJ/mol) governing the CO librations.     

Isolation and sequence studies of cysteinyl peptides from Spirulina glutathione reductase: comparison of active site cysteine peptides with those of other flavoprotein disulfide oxidoreductases

The amino acid sequences of the cysteinyl peptides of Spirulina sp. glutathione reductase were determined. Spirulina glutathione reductase was covalently bound to Thiopropyl-Sepharose 6B in the presence of 8M urea through thiol-disulfide exchange. After tryptic digestion, 4 distinct cysteinyl peptides were finally isolated from NADPH-reduced glutathione reductase and 2 from oxidized glutathione reductase. The amino acid sequences of the two cysteinyl peptides which could not be isolated from the oxidized glutathione reductase were very similar to those around the active site disulfide of the other flavoprotein disulfide oxidoreductases and a unique replacement of asparagine and valine by isoleucine and arginine between the two cysteine residues was found. The other two peptides isolated from both oxidized and reduced glutathione reductase also show considerable homology to the corresponding parts of human and Escherichia coli glutathione reductases.     

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.     

The glutaredoxin -C-P-Y-C- motif: influence of peripheral residues

The variety of cellular functions performed by proteins of the thioredoxin superfamily is made possible by the wide range of redox potential associated with their active site -Cys-X-X-Cys- motif. The determinants of these differences in redox potential are of considerable interest but are not well understood. E. coli Glutaredoxin 1 (Grx1) and 3 (Grx3) are important model systems with different redox properties, despite sharing the same -Cys-Pro-Tyr-Cys- motif, very similar overall structures, and 33% sequence identity. Very long molecular dynamics simulations (0.25 micros total) and electrostatic calculations provide a revised view of the reduced Grx1 active site, which now can be reconciled with biochemical and functional data. Comparison of this new model to Grx3 uncovers differences in the structure, dynamics, and electrostatics of these active sites. The influence of peripheral residues on the properties of the -Cys-X-X-Cys- motif is illustrated specifically with the effect of a Lys to Arg substitution.     

Active site of trypanothione reductase. A target for rational drug design.

The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.