The active site region plays a critical role in Na+ binding to thrombin

The catalytic activity of thrombin and other enzymes of the blood coagulation and complement cascades is enhanced significantly by binding of Na⁺ to a site >15 Å away from the catalytic residue S195, buried within the 180 and 220 loops that also contribute to the primary specificity of the enzyme. Rapid kinetics support a binding mechanism of conformational selection where the Na⁺-binding site is in equilibrium between open (N) and closed (N^∗) forms and the cation binds selectively to the N form. Allosteric transduction of this binding step produces enhanced catalytic activity. Molecular details on how Na⁺ gains access to this site and communicates allosterically with the active site remain poorly defined. In this study, we show that the rate of the N∗→N transition is strongly correlated with the analogous E∗→E transition that governs the interaction of synthetic and physiologic substrates with the active site. This correlation supports the active site as the likely point of entry for Na⁺ to its binding site. Mutagenesis and structural data rule out an alternative path through the pore defined by the 180 and 220 loops. We suggest that the active site communicates allosterically with the Na⁺ site through a network of H-bonded water molecules that embeds the primary specificity pocket. Perturbation of the mobility of S195 and its H-bonding capabilities alters interaction with this network and influences the kinetics of Na⁺ binding and allosteric transduction. These findings have general mechanistic relevance for Na⁺-activated proteases and allosteric enzymes.     

Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

Abstract

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G₃T₂G₃TGTG₃T₂G₃) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I₂ to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction.

Communicated by Ramaswamy H. Sarma     

Rapid kinetics of Na+ binding to thrombin

The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E(*) and E, where E(*) is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E(*) transition. As a result, under conditions of physiologic temperature and salt concentrations, the E(*) form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.     

Molecular dissection of Na+ binding to thrombin

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.     

Conserved water molecules in the specificity pocket of serine proteases and the molecular mechanism of Na+ binding

Conservation of clusters of buried water molecules is a structural motif present throughout the serine protease family. Frequently, these clusters are shaped as water channels forming extensive hydrogen-bonding networks linked to the protein backbone. The most conspicuous example is the water channel present in the specificity pocket of trypsin and thrombin. In thrombin, other vitamin K-dependent proteases, and some complement factors, Na⁺ binds in this water channel and enhances allosterically the catalytic activity of the enzyme, whereas digestive and fibrinolytic proteases are devoid of such regulation. A comparative analysis of proteases with and without Na⁺ binding capability reveals the role of the water channel in maintaining the structural organization of the specificity pocket and in Na⁺ coordination. This enables the formulation of a molecular mechanism for Na⁺ binding in thrombin and leads to the identification of the structural changes necessary to engineer a functional Na⁺ site and enhanced catalytic activity in trypsin and other proteases. Proteins 30:34–42, 1998. © 1998 Wiley-Liss, Inc.     

The pha2 gene cluster involved in Na+ resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter

Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.     

Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1

We have determined the structure of the archaeal sodium/proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane‐spanning α‐helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six‐helix bundle at the tip of the protomer is conserved. The six‐helix bundle of NhaA contains two partially unwound α‐helices thought to harbour the ion‐translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N‐terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed.     

Rigidification of the autolysis loop enhances Na(+) binding to thrombin

Binding of Na⁺ to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na⁺ is weak due to large heat capacity and enthalpy changes associated with binding, and the K_d = 80 mM ensures only 64% saturation of the site at the concentration of Na⁺ in the blood (140 mM). Residues controlling Na⁺ binding and activation have been identified. Yet, attempts to improve the interaction of Na⁺ with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na⁺ affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na⁺ binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes.     

The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.

The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.     

Binding of Na+ ions to the Na,K-ATPase increases the reactivity of an essential residue in the ATP binding domain

Treatment of the canine renal Na,K-ATPase with N-(2-nitro-4-isothiocyanophenyl)-imidazole (NIPI), a new imidazole-based probe, results in irreversible loss of enzymatic activity. Inactivation of 95% of the Na,K-ATPase activity is achieved by the covalent binding of 1 molecule of [3H]NIPI to a single site on the alpha-subunit of the Na,K-ATPase. The reactivity of this site toward NIPI is about 10-fold greater when the enzyme is in the E1Na or sodium-bound form than when it is in the E2K or potassium-bound form. K+ ions prevent the enhanced reactivity associated with Na+ binding. Labeling and inactivation of the enzyme is prevented by the simultaneous presence of ATP or ADP (but not by AMP). The apparent affinity with which ATP prevents the inactivation by NIPI at pH 8.5 is increased from 30 to 3 microM by the presence of Na+ ions. This suggests that the affinity with which native enzyme binds ATP (or ADP) at this pH is enhanced by Na+ binding to the enzyme. Modification of the single sodium-responsive residue on the alpha-subunit of the Na,K-ATPase results in loss of high affinity ATP binding, without affecting phosphorylation from Pi. Modification with NIPI probably alters the adenosine binding region without affecting the region close to the phosphorylated carboxyl residue aspartate 369. Tightly bound (or occluded) Rb+ ions are not displaced by ATP (4 mM) in the inactivated enzyme. Thus modification of a single residue simultaneously blocks ATP acting with either high or low affinity on the Na,K-ATPase. These observations suggest that there is a single residue on the alpha-subunit (probably a lysine) which drastically alters its reactivity as Na+ binds to the enzyme. This lysine residue is essential for catalytic activity and is prevented from reacting with NIPI when ATP binds to the enzyme. Thus, the essential lysine residue involved may be part of the ATP binding domain of the Na,K-ATPase.     

The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding

The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (deltaK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (alpha-NAPAP) to the wild-type (WT) and deltaK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free deltaK9 and WT thrombin, respectively. Urea-induced denaturation studies showed higher instability of the deltaK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A-B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.     

Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP

To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain. Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding. The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation. The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP [K(1/2)(ATP) 12 nM]. Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol. Mutations of Cys(549) did not affect ATP binding. In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).     

Thermodynamics of protein‐cation interaction: Ca+2 and Mg+2 binding to the fifth binding module of the LDL receptor

The ligand binding domain of the LDL receptor (LDLR) contains seven structurally homologous repeats. The fifth repeat (LR5) is considered to be the main module responsible for the binding of lipoproteins LDL and β‐VLDL. LR5, like the other homologous repeats, is around 40‐residue long and contains three disulfide bonds and a conserved cluster of negatively charged residues surrounding a hexacoordinated calcium ion. The calcium coordinating cage is formed by the backbone oxygens of W193 and D198, and side‐chain atoms of D196, D200, D206, and E207. The functionality of LDLR is closely associated with the presence of calcium. Magnesium ions are to some extent similar to calcium ions. However, they appear to be involved in different physiological events and their concentrations in extracellular and intracellular compartments are regulated by different mechanisms. Whether magnesium ions can play a role in the complex cycle of LDLR internalization and recycling is not known. We report here a detailed study of the interaction between LR5 and these two cations combining ITC, emission fluorescence, high resolution NMR, and MD simulations, at extracellular and endosomal pHs. Our results indicate that the conformational stability and internal dynamics of LR5 are strongly modulated by the specific bound cation. It appears that the difference in binding affinity for these cations is somewhat compensated by their different concentrations in late LDL‐associated endosomes. While the mildly acidic and calcium‐depleted environment in late endosomes has been proposed to contribute significantly to LDL release, the presence of magnesium might assist in efficient LDLR recycling. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: Selectivity and kinetic properties of ion binding sites

Summary The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [³H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na⁺ and K⁺ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The K _m for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na⁺ and K⁺ binding sites. Na⁺ and K⁺ were partially replaceable, with the selectivity of the Na⁺ site being Na⁺ > Li⁺ > NH ₄ ⁺ ; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ also supported a small amount of specific binding when substituted for Na⁺. The selectivity of the K⁺ site was K⁺ Rb⁺ > NH ₄ ⁺ > Cs⁺; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li⁺ could partially substitute for Na⁺ in supporting coteansport, but other monovalent cations were completely inactive. The order of potency at the K⁺ site was NH ₄ ⁺ > K⁺ Rb⁺ > Cs⁺ other monovalent cations. The effect of Cl^- on bumetanide binding was biphasic, being stimulatory at low [Cl^-] but inhibitory at high [Cl^-]. As this implies the existence of two Cl^- binding sites (termed Cl _H and Cl _L for the high- and low- affinity sites, respectively) each phase was examined individually. Cl^- binding to Cl _H could be described by a rectangular hyperbola with a K _m of 2.5 mm, while kinetic analysis of the inhibition of bumetanide binding at high [Cl^-] revealed that it was of a noncompetitive type (K _i = 112.9 mm). The selectivity of anion binding to the two sites was distinct. Cl _H was highly selective with Cl^- > SCN^- > Br^-; F^-, NO ₃ ^- , ClO ₄ ^- , MeSO ₄ ^- , gluconate^- and SO ₄ ^2- were inactive. The efficacy of anion inhibition of binding to Cl _L was ClO ₄ ^- > I^- > SCN^- > NO₃ > Cl^-; F^-, MeSO ₄ ^- , gluconate^-, and SO ₄ ^2- were inactive. Thus, Cl _H is much more selective than Cl _L and largely accounts for the specificity of the system with respect to anion transport. SO ₄ ^- , NO ₃ ^- , I^-, SCN^- and ClO ₄ ^- did not support cotransport when bound to Cl _L and the latter three anions were inhibitory. Mg²⁺ was found to stimulate binding at a narrowly defined peak around 1.5 mm, but was inhibitory at higher concentrations. Other divalent cations caused a similar inhibition of bumetanide binding but did not exert a stimulatory effect at 1.5 mm. Divalent cations have little effect on cotransport in intact cells at concentrations up to 20 mm, suggesting that their effects on diuretic binding reflect interactions at internally disposed sites. Bumetanide binding was optimal at a pH of 7.8–8.1 and declined sharply as the pH was lowered towards 6. The titration curve correlated well with the effect of pH on cotransport in intact cells; the inhibitory effect of low pH suggests that protonation of the cotransporter may inhibit its function.We thank Drs. Brad Pewitt, John Westley and Mrinalini Rao for discussion, Sara Leung and Artelia Watson for their excellent technical assistance, and Dr. R.J. Turner for his gift of [³H] bumetanide. This work was supported in part by Cystic Fibrosis Center grant #CF RO11 7-04.     

Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

TLR4 polymorphisms such as Asp299Gly and Thr399Ile related to Gram-negative sepsis have been reported to result in significantly blunted responsiveness to LPS. Our study group previously screened other TLR4 polymorphic variants by checking the NF-κB activation in comparison to wild type (WT) TLR4 in human embryonic kidney 293T cells. In this study, we found that the Lys694Arg (K694R) polymorphism reduced the activation of NF-κB, and the production of downstream inflammatory factors IL-1, TNF-α and IL-6, representing the K694R polymorphism, led to blunted responsiveness to LPS. Then, we examined the influence of the K694R polymorphism on total and cell-surface TLR4 expression by Western blotting and flow cytometry, respectively, but observed no differences between the K694R polymorphism and WT TLR4. We also used co-immunoprecipitation to determine the interaction of the K694R polymorphism and WT TLR4 with their co-receptor myeloid differentiation factor 2 (MD2) and their downstream signal adaptor MyD88. We found that K694R reduced the recruitment of MyD88 in TLR4 signalling but had no impact on the interaction with MD2.     

Characterization of fatty acid interaction with ouabain and vanadate binding to (Na+ + K+)-activated ATPase

The candidateship of unsaturated fatty acids as endogenous ouabain-like factors was studied. Binding of the artificial ligand vanadate at the intracellular phosphorylation epitope of membrane-bound Na+/K+-ATPase was unaffected by linoleic and arachidonic acid. In the (Mg2+ + Pi)-facilitated system for ouabain binding they were characterized as noncompetitive inhibitors of cardiac glycoside binding, however. The ouabain binding capacity as well as the affinity decreased and the ouabain dissociation rate was accelerated by fatty acids. In the presence of vanadate for facilitation of ouabain binding an increase in ouabain affinity was seen. It is concluded that elementary criteria for the characterization of unsaturated fatty acids as ouabain-like factors are not fulfilled. The ratio between E2-subconformations of Na+/K+-ATPase with different ouabain affinities may be changed by incorporation of fatty acids in the lipid membrane.     

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near — 60mV, yet are scarcely depolarized by 80 mM Rb⁺ and are relatively impermeable to ⁸⁶Rb⁺. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K⁺ to cells suspended in a K⁺ free medium, or of Na⁺ to cells in a Na⁺ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na⁺, K⁺ and H⁺ which can function as Na⁺:K⁺ and Na⁺:H⁺ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na⁺ and K⁺, is produced by an electrogenic Na⁺ pump sustained by electroneutral exchanges, and restricted by anion leakage.     

The role of Li+, Na+, and K+ in the ligand binding inside the human acetylcholinesterase gorge

Alkali cations can affect the catalytic efficiency of enzymes. This is particularly true when dealing with enzymes whose substrate bears a formal positive charge. Computational and biochemical approaches have been combined to shed light on the atomic aspects of the role of Li(+), Na(+), and K(+) on human acetylcholinesterase (hAChE) ligand binding. In this respect, molecular dynamics simulations and our recently developed metadynamics method were applied to study the entrance of the three cations in the gorge of hAChE, and their effect on the dynamical motion of a ligand (tetramethylammonium) from the bulk of the solvent into the deep narrow enzyme gorge. Furthermore, in order to support the theoretical results, K(M) and k(cat) for the acetylcholine hydrolysis in the presence of the three cations were evaluated by using an approach based on the Ellman's method. The combination of computational and biochemical experiments clearly showed that Li(+), Na(+), and K(+) may influence the ligand binding at the hAChE gorge.