Assignment of the histidine 12-threonine 45 hydrogen-bonded proton in the NMR spectrum of ribonuclease A in H2O.

Described herein are proton nmr experiments on chemically modified derivatives of ribonuclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pK_a of 6.1 in H₂O solution. Histidines 48 and 105, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate–water solutions. This exchangeable resonance titrates with modified pK_a's and constant area over the above pH range in His-119-N₁-carboxymethylated-RNase A and des-(121–124)-RNase A, thus eliminating the imidazole N₃ proton in the His 119-Asp 121 hydrogen bond. In His-12-N₁-carboxymethylated-RNase A, this resonance is also observable, but broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH-independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidazole N₁ proton of His 12, which is hydrogen-bonded to the carbonyl oxygen of Thr 45 in the crystal.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

Proton NMR investigation of heme and surrounding proton in low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla

¹H NMR spectra of low-spin cyanide-ligated bacterial hemoglobin fromVitreoscilla (VtHb-CN) are reported. The assignments of the¹H NMR spectra of VtHb-CN have been made through MCOSY, NOESY, 1D TOE and SUPERWEFT experiments. Almost all resonance peaks of heme and ligated His85 are identified. The spin-lattice relaxation timeT ¹’s and the variation relationships of chemical shifts of these peaks with temperature have been acquired, from which the distances between the measured protons and Fe³⁺, and the diamagnetic chemical shifts have been acquired, respectively. The ionization constants of pK _a’s of ligated His85 are determined through pH titration of chemical shift, which is 4.95 for ligated His85 C₂H proton. The lower pK _a is attributed to the influence of the Fe³⁺ of carrying positive charge and the coordination of His85 and Fe³⁺ of heme.     

Histidine proton resonances of carbonmonoxyhaemoglobins A and Cowtown in chloride-free buffer

A re-examination of the C-2 histidine proton resonances of haemoglobins A and Cowtown (His HC3(146)β → Leu) in chloride-free Hepes buffer has shown that all the resonances present in haemoglobin A are present in haemoglobin Cowtown, so that the pK_a of His HC3(146)β cannot be determined by nuclear magnetic resonance in this buffer.     

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.     

The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices

Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 m-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146)β. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94)β, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 m-NaCl + 0.2 m-phosphate this change of ligation lowered the pK_a of the histidine from 8.0 in Hb³ to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pK_a in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect.We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)β → Leu), HbCO Wood (His FG4(97)β → Leu) and HbCO Malmø (His FG4(97)β → Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)β in fact belongs to His FG4(97)β. Although in Hb the pK_a of His HC3(146)β is 8.05 ± 0.05 independent of ionic strength, in HbCO its pK_a drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pK_a of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973).In HbCO A, His FG4(97)β does not contribute to the Bohr effect, but in HbCO from which His HC3(146)β has been cleaved (HbCO des-His), His FG4(97)β is in equilibrium between two conformations with different pK_a values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety.In HbCO A, His FG4(97)β has a pK_a of 7.8 compared to the pK_a value of about 6.6 characteristic of free histidines at the surface of proteins. This high pK_a is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of α-helices, which manifests itself even when they lie on the surface of the protein.     

Synthesis of N-acridinyl-N'-alkylguanidines: dramatic influence of amine to guanidine replacement on the physicochemical properties

Transformation of aminoacridines into N-acridinyl-N′-alkylguanidines is described. The chosen procedure allows introduction of pendent substituents (exemplified by N,N-dimethylaminopropyl chain) into key acridinyl thioureas, thus opening the way to structural diversity. Spectroscopic study and pK_a determination show that the presence of the strongly basic guanidine has a dramatic influence on the ionization of the acridine nucleus by lowering the pk_a value down to 4.49.     

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.     

Elucidating Relayed Proton Transfer through a His–Trp–His Triad of a Transmembrane Proton Channel by Solid-State NMR

Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H¹⁹xxxW²³xxxH²⁷ motif. The proton-dissociation constants (pK_a's) of H19 in BM2 were found to be much lower than the pK_a's of H37 in AM2. To determine if the lower pK_a's result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. ¹⁵N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pK_a's of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation–π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.     

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK_a values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK_a values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK_a values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK_a was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N^ε2 state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK_a calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK_a values are governed by short-range polar and Coulomb interactions.     

Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase

The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the α class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pK_a for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pK_a between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in ΔpK_a; however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase.     

Transition-state structure for a conformation change of ribonuclease

The rate constants k₁₂ⁿ for isomerization of the E₁H isomer (pK_a 8 in H₂O) of ribonuclease-A to the E₂H isomer (pK_a = 6.1 in H₂O), determined from proton-uptake measurements by the temperature-jump technique, in mixtures of protium and deuterium oxides (atom fraction of deuterium n), are described by the equation k₁₂ⁿ = (733 ± 16)(1 − n + [0.46 ± 0.04]n)(1 − n + 0.69n)²sec⁻¹ at 25°C. On the basis of the absolute magnitude of the rate constant, the magnitude of the solvent isotope effect and the proton inventory, it appears that the rate-determining step is proton transfer to a water molecule from the imidazolium form of a histidine residue, with a product-like activated complex resembling a hydronium ion. The subsequent motion of the protein structure to generate the new isomer (conformation change) must then occur in a time approaching a vibrational period. Alternative but less likely mechanisms include rate-limiting protein reorganization concerted with proton transfer to water, rate-limiting diffusion of hydronium ion away from the enzyme, or “solvation catalysis” of protein reorganization.     

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

His349 in human transferrin (hTF) is a residue critical to transferrin receptor (TFR)-stimulated iron release from the C-lobe. To evaluate the importance of His349 on the TFR interaction, it was replaced by alanine, aspartate, lysine, leucine, tryptophan, and tyrosine in a monoferric C-lobe hTF construct (Fe_ChTF). Using a stopped-flow spectrofluorimeter, we determined rate processes assigned to iron release and conformational events (in the presence and in the absence of the TFR). Significantly, all mutant/TFR complexes feature dampened iron release rates. The critical contribution of His349 is most convincingly revealed by analysis of the kinetics as a function of pH (5.6–6.2). The Fe_ChTF/TFR complex titrates with a pK _a of approximately 5.9. By contrast, the H349A mutant/TFR complex releases iron at higher pH with a profile that is almost the inverse of that of the control complex. At the putative endosomal pH of 5.6 (in the presence of salt and chelator), iron is released from the H349W mutant/TFR and H349Y mutant/TFR complexes with a single rate constant similar to the iron release rate constant for the control; this suggests that these substitutions bypass the required pH-induced conformational change allowing the C-lobe to directly interact with the TFR to release iron. The H349K mutant proves that although the positive charge is crucial to complete iron release, the geometry at this position is also critical. The H349D mutant shows that a negative charge precludes complete iron release at pH 5.6 both in the presence and in the absence of the TFR. Thus, histidine uniquely drives the pH-induced conformational change in the C-lobe required for TFR interaction, which in turn promotes iron release.     

Steric Enhancement of Imidazole Basicity incis-Urocanic Acid Derivatives: Models for the Action of Chymotrypsin

To test the hypothesis that substrate-induced steric compression between His 57 and Asp 102 at the active site of chymotrypsin can increase the basicity of His 57, we have synthesized thecis- andtrans-isomers of 2-bromo-3-(N-tritylimidazole)-2-propenoic acid and 2-chloro-3-(N-tritylimidazole)-2-propenoic acid and compared selected properties with those ofcis-andtrans-urocanic acids. Thecis-isomers display low field¹H NMR signals at 17 ppm in dimethylsulfoxide, similar tocis-urocanic acid; whereas thetrans-isomers do not show strong hydrogen bonds. Increasing the size of the C2 substituent (H < Cl < Br) in thecis-isomers increases the pK_aof the imidazolium group from 6.78 for H to 7.81 and 9.10 for Cl and Br, respectively; whereas the pK_as of thetransisomers are all 6.0 ± 0.1. The results indicate that thecis-urocanic acid derivatives with large substituents at C2 act as proton sponges in water, and they support the concept that steric compression in the catalytic triad of chymotrypsin can increase the basicity of His 57.     

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.     

Mechanistic Insight into the H<Subscript>2</Subscript>O/D<Subscript>2</Subscript>O Isotope Effect in the Proton Transport of the Influenza Virus M2 Protein

The M2 proton channel is essential for the replication of the flu virus and is a known drug target. The functional mechanism of channel activation and conductance is key to both the basic biology of viral replication and the design of drugs that can withstand mutations. A quantitative model was previously developed for calculating the rate of proton transport through the M2 channel. The permeant proton was assumed to diffuse to the pore, obligatorily bind to the His37 tetrad, and then dissociate and be released to either side of the tetrad. Here the model is used to calculate the effect of a change in solvent from H₂O to D₂O on the rate of proton transport. The solvent substitution affects two parameters in the model: the proton diffusion constant and the pK _a for proton binding to the His37 tetrad. When the known effects on these two parameters are included, the deuterium isotope effect calculated from the model is in quantitatively agreement with experimental results. This strict test of the theoretical model provides strong support for the hypothesis that the permeant proton obligatorily binds to and then unbinds from the His37 tetrad. This putatively essential role of the His37 tetrad in the functional mechanism of the M2 channel makes it a promising target for designing mutation-tolerant drugs.     

Effects of 3′-C-Methylation on the Hydrolytic Stability and Hydroxyl pK a Values of Dinucleoside 2′,5′- and 3′,5′-Monophosphates

Abstract

The first-order rate constants for hydrolysis of 3′-C-methyluridylyl(2′,5′)- and -(3′,5′)adenosine and the corresponding native dinucleoside monophosphates (2′,5′- and 3′,5′-UpA) have been determined as a function of hydroxide-ion concentration (0.025 - 7 M) at 25°C. In addition to the effects on the hydrolytic stability of the compounds, the effects of the 3′-C-methyl substitution on the kinetically determined pK _a values for the sugar hydroxyls of the undine moiety are discussed.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.