Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Toward the Development of Metal‐Free Synthetic Nucleases: Cleavage of a Model Substrates by 1,4‐Diazabicyclo[2.2.2]Octane Derivatives

Artificial ribonucleases of A_nBCL series were synthesized by solid‐phase method. They consist of a hydrophobic alkyl radical A (n = 3–12 carbon atoms), an “RNA‐binding domain” B (bisquaternary salt of 1,4‐diazabicyclo[2.2.2]octane), a “catalytic domain” C (histidine residue) and a “linker” L that joins the domains B and C. The effect of the alkyl radical on the catalytic properties of the chemical catalyst was studied using three activated phosphate ester substrates: p‐nitrophenyl phosphate, bis‐p‐nitrophenyl phosphate, and thymidine‐3′‐p‐nitrophenyl phosphate.     

1H nuclear magnetic resonance studies of histidine-containing di- and tripeptides. Estimation of the effects of charged groups on the pKa value of the imidazole ring.

The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pK_a values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pK_a value of the imidazole ring in N-acetyl-L -histidine methylamide was assumed to represent the intrinsic (or unperturbed) pK_a of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pK_a values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO₂^? and NH₃⁺ groups located at various positions. Some other factors affecting the pK_a value of the imidazole ring are also discussed.     

Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model

One of the most popular and simple models for the calculation of pK_as from a protein structure is the semi‐macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK_as. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK_as; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK_as. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK_a values (where the calculation should reproduce the pK_a within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK_a in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK_a should be shifted, and validation of force field parameters for cysteine residues. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Role of Lys281 in the <Emphasis Type="Italic">Dunaliella salina</Emphasis> (6-4) Photolyase Reaction

His³⁵⁴ and His³⁵⁸, two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His⁴⁰¹ and His⁴⁰⁵, in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys²⁸¹ might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys²⁸¹, which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His⁴⁰¹ when the environmental pH is higher than the pKa value of histidine.     

An abnormal pK(a) value of internal histidine of the insulin molecule revealed by neutron crystallographic analysis

Insulin is stored in pancreatic β-cell as hexameric form with Zn²⁺ ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn²⁺ ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn²⁺ free insulin at pD6.6 and pD9 by neutron crystallographic analyses. HisB10 is doubly protonated not only at pD6.6 but also at pD9, indicating an abnormal pK_a of this histidine. It is suggested that HisB10 acts on a strong cation capture and contributes to the high stability of the hexameric form in pancreas.     

The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the pK_a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK_a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low pK_a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK_a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine pK_a. However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine pK_a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK_a. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the pK_a of the nucleophilic cysteine in NrdH‐redoxins.     

Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583

Aims: To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. Methods and Results: The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C‐terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4‐nitrophenyl N,N′‐diacetyl‐β‐d ‐chitobioside with K_m values of 1·6 and 2·1 mmol l^?1, respectively, and k_cat values of 21·6 and 6·5 s^?1. The enzymes also showed activity towards 4‐nitrophenyl β‐d ‐N, N′, N″‐triacetylchitotriose and carboxy‐methyl‐chitin‐Remazol Brilliant Violet but not towards 4‐nitrophenyl N‐acetyl‐β‐d ‐glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4‐nitrophenyl β‐d ‐cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. Conclusion: The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. Significance and Impact of the Study: This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food‐manufacturing plants.     

Histidine Residues in the Na+-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function,Modulating pH Sensitivity,Transporter Kinetics,Na+ Cooperativity,Conformational Stability,and Subcellular Localization

Na⁺-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His¹⁰⁹, His²⁰³, His²⁰⁶, His²⁶⁹, and His⁴¹³, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na⁺ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His⁴¹³, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na⁺ and loss of Na⁺ cooperativity, which leads to a decreased V_max without altering the transport K_m; (ii) exofacial histidine residues His²⁰³, His²⁰⁶, and His⁴¹³ may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K_m; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.     

Diethylpyrocarbonate reactivity ofKlebsiella aerogenes urease: Effect ofpH and active site ligands on the rate of inactivation

Reaction ofKlebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. ThepH dependence for the rate of inactivation indicated that the reactive residue possessed apK _a of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.     

Origin of the pKa shift of the catalytic lysine in acetoacetate decarboxylase

The pK_a value of Lys115, the catalytic residue in acetoacetate decarboxylate, was calculated using atomic coordinates of the X-ray crystal structure with consideration of the protonation states of all titratable sites in the protein. The calculated pK_a value of Lys115 (pK_a(Lys115)) was unusually low (≈6) in agreement with the experimentally measured value. Although charged residues impact pK_a(Lys115) considerably in the native protein, the significant pK_a(Lys115) downshift in the protein with respect to aqueous solution was mainly due to loss of the solvation energy in the catalytic active site relative to bulk water.     

pH dependence of conformational fluctuations of the protein backbone

Proton binding equilibria (pK_a values) of ionizable groups in proteins are exquisitely sensitive to their microenvironments. Apparent pK_a values measured for individual ionizable residues with NMR spectroscopy are actually population‐weighted averages of the pK_a in different conformational microstates. NMR spectroscopy experiments with staphylococcal nuclease were used to test the hypothesis that pK_a values of surface Glu and Asp residues are affected by pH‐sensitive fluctuations of the backbone between folded and locally unfolded conformations. ¹⁵N spin relaxation studies showed that as the pH decreases from the neutral into the acidic range the amplitudes of backbone fluctuations in the ps‐ns timescale increase near carboxylic residues. Hydrogen exchange experiments suggested that backbone conformational fluctuations promoted by decreasing pH also reflect slower local or sub‐global unfolding near carboxylic groups. This study has implications for structure‐based pK_a calculations: (1) The timescale of the backbone's response to ionization events in proteins can range from ps to ms, and even longer; (2) pH‐sensitive fluctuations of the backbone can be localized to both the segment the ionizable residue is attached to or the one that occludes the ionizable group; (3) Structural perturbations are not necessarily propagated through Coulomb interactions; instead, local fluctuations appear to be coupled through the co‐operativity inherent to elements of secondary structure and to networks of hydrogen bonds. These results are consistent with the idea that local conformational fluctuations and stabilities are important determinants of apparent pK_a values of ionizable residues in proteins. Proteins 2014; 82:3132–3143. © 2014 Wiley Periodicals, Inc.     

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase

Background

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK _a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.

Methodology/Principal Findings

Using His-HDX-MS, the pK _a values and the half-lives (t _1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP⁺ (DHFR-folate-NADP⁺) were determined. The results showed that the two parameters (pK _a and t _1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK _a, t _1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK _a and t _1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP⁺. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.

Conclusions/Significance

Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Predicting protein pKa by environment similarity

A statistical method to predict protein pK_a has been developed by using the 3D structure of a protein and a database of 434 experimental protein pK_a values. Each pK_a in the database is associated with a fingerprint that describes the chemical environment around an ionizable residue. A computational tool, MoKaBio, has been developed to identify automatically ionizable residues in a protein, generate fingerprints that describe the chemical environment around such residues, and predict pK_a from the experimental pK_a values in the database by using a similarity metric. The method, which retrieved the pK_a of 429 of the 434 ionizable sites in the database correctly, was crossvalidated by leave‐one‐out and yielded root mean square error (RMSE) = 0.95, a result that is superior to that obtained by using the Null Model (RMSE 1.07) and other well‐established protein pK_a prediction tools. This novel approach is suitable to rationalize protein pK_a by comparing the region around the ionizable site with similar regions whose ionizable site pK_a is known. The pK_a of residues that have a unique environment not represented in the training set cannot be predicted accurately, however, the method offers the advantage of being trainable to increase its predictive power. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

The structure of carbonyl horseradish peroxidase: spectroscopic and kinetic characterization of the carbon monoxide complexes of His-42 → Leu and Arg-38 → Leu mutants

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pK _as at 7.4 and 4.0. Data obtained with the His-42?→?Leu [(H42L)HRPC*)] and Arg-38?→?Leu [(R38L)HRPC*] mutants allowed the pK _a at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν?(CO) bands at 1934?cm^–1 and 1905?cm^–1 whose relative intensity changed with pH, showing an acidic and a basic pK _a as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118?:?3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2?cm^–1 (pH?7.0) and 1941.5?cm^–1 (pH?5.0) respectively. Acidic and alkaline pK _as were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Söret maxima. (H42L)HRPC*-CO exhibited a pK _a at ～pH?4.0 but no alkaline pK _a. (R38L)HRPC*-CO exhibited a single pK _a at pH?6.5. Shifts of 2–3?cm^–1 in ν?(CO) with (H42L)HRPC*-CO in D₂O show that a distal residue is H-bonding to the CO in this variant at both pD?7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν?(CO) band was observed at pD?5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH?8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pK _a between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.     

Ionization constants and thermodynamic parameters of histidine and derivatives

The pK_a values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pK_a values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pK_a in histidine and its studied derivatives. The N^τ-H tautomer is favored over the N^π-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.     

Mutation of non-conserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase

Improvement of enzyme function by engineering pH dependence of enzymatic activity is of importance for industrial application of Bacillus circulans xylanases. Target mutation sites were selected by structural alignment between B. circulans xylanase and other xylanases having different pH optima. We selected non-conserved mutant sites within 8 Å from the catalytic residues, to see whether these residues have some role in modulating pK_as of the catalytic residues. We hypothesized that the non-conserved residues which may not have any role in enzyme catalysis might perturb pK_as of the catalytic residues. Change in pK_a of a titratable group due to change in electrostatic potential of a mutation was calculated and the change in pH optimum was predicted from the change in pK_a of the catalytic residues. Our strategy is proved to be useful in selection of promising mutants to shift the pH optimum of the xylanases towards desired side.     

Flavin oxidation in flavin‐dependent N‐monooxygenases

Siderophore A (SidA) from Aspergillus fumigatus is a flavin‐containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H₂O₂ is the main product. The effect of pH on steady‐state kinetic parameters was measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pK_a value >9 was determined and assigned to the FAD‐N5 atom. In the presence of Orn, the pH dependence displayed a pK_a value of 6.7 ±0.1 and of 7.70 ±0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a‐hydroperoxyflavin (FAD_OOH). Flavin oxidation with Q102A showed a pK_a value of ~8.0. The data are consistent with the pK_a of the FAD N5‐atom being modulated to a value >9 in the absence of Orn, which aids in the stabilization of FAD_OOH. Changes in the FAD‐N5 environment lead to a decrease in the pK_a value, which facilitates elimination of H₂O₂ or H₂O. These findings are supported by solvent kinetic isotope effect experiments, which show that proton transfer from the FAD N5‐atom is rate limiting in the absence of a substrate, however, is significantly less rate limiting in the presence of Orn and or Lys.