Role of Gly117 in the cation/melibiose symport of MelB of Salmonella typhimurium

The melibiose permease of Salmonella typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+), and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na(+)-binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding of cosubstrates depended on the physical-chemical properties of the side chain. Compared with WT MelB(St), the Gly117 → Ala mutant exhibited little difference in either cosubstrate binding or stimulation of melibiose transport by Na(+) or Li(+), but all other mutations reduced melibiose active transport and efflux, and decreased the apparent affinity for Na(+). The bulky Trp at position 117 caused the greatest inhibition of melibiose binding, and Gly117 → Arg yielded less than a 4-fold decrease in the apparent affinity for melibiose at saturating Na(+) or Li(+) concentration. Remarkably, the mutant Gly117 → Arg catalyzed melibiose exchange in the presence of Na(+) or Li(+), but did not catalyze melibiose translocation involving net flux of the coupling cation, indicating that sugar is released prior to release of the coupling cation. Taken together, the findings are consistent with the notion that Gly117 plays an important role in cation binding and translocation.     

Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ～1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ～1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ～500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.     

Evidence for intraprotein charge transfer during the transport activity of the melibiose permease from Escherichia coli.

Electrogenic activity associated with the activity of the melibiose permease (MelB) of Escherichia coli was investigated by using proteoliposomes containing purified MelB adsorbed onto a solid-supported membrane. Transient currents were selectively recorded by applying concentration jumps of Na+ ions (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-methyl galactoside, raffinose) using a fast-flow solution exchange system. Characteristically, the transient current response was fast, including a single decay exponential component (tau approximately 15 ms) on applying a Na+ (or Li+) concentration jump in the absence of sugar. On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the cation and sugar substrates, the electrical transients were biphasic and comprised both the fast and an additional slow (tau approximately 350 ms) decay components. Finally, selective inactivation of the cosubstrate translocation step by acylation of MelB cysteins with N-ethyl maleimide suppressed the slow response components and had no effect on the fast transient one. We suggest that the fast transient response reflects charge transfer within MelB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane. From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s(-1) and one for melibiose binding in the absence of Na+ of k approximately 10 s(-1).     

Sugar binding induced charge translocation in the melibiose permease from Escherichia coli

Electrogenic events associated with the activity of the melibiose permease (MelB), a transporter from Escherichia coli, were investigated. Proteoliposomes containing purified MelB were adsorbed to a solid supported lipid membrane, activated by a substrate concentration jump, and transient currents were measured. When the transporter was preincubated with Na(+) at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed. This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein. Further analysis showed that the charge displacement is neither related to extra Na(+) binding to the transporter, nor to the displacement of already bound Na(+) within the transporter. The electrogenic melibiose binding process is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles. A kinetic model is suggested, in which Na(+) and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement. These binding reactions are fast in the presence of the respective cosubstrate (k > 50 s(-1)).     

Changes in secondary structures and acidic side chains of melibiose permease upon cosubstrates binding

Infrared difference spectroscopy analysis of the purified melibiose permease of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na(+), melibiose) in either H(2)O or in D(2)O media. Essentially, the data first show that addition of Na(+) induces appearance of peaks assigned to changes in the environment and/or orientation of alpha-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na(+) produces peaks corresponding to additional changes of alpha-helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+) cm(-1)/1576 (-) cm(-1)) appearing in the Na(+)-induced difference spectrum is assigned to the antisymmetric stretching of COO(-) groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na(+) co-ordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (-) cm(-1)/1567 (+) cm(-1)), appear in the melibiose-induced difference spectra, and may again be assigned to COO(-) groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding.     

Study of amide-proton exchange of Escherichia coli melibiose permease by attenuated total reflection-Fourier transform infrared spectroscopy: evidence of structure modulation by substrate binding.

The accessibility of Escherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H(+), Na(+), melibiose). Analysis of the amide II band upon (2)H(2)O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H(+)*melibiose*MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on beta-sheet accessibility, with the complex H(+)*melibiose*MelB being the most protected state against exchange, followed by Na(+)*melibiose*MelB. Although of smaller magnitude, similar changes in alpha-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly beta-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4-5 and 10-11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized.     

Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions

An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.     

Three-dimensional structure of the sugar symporter melibiose permease from cryo-electron microscopy

Melibiose permease (MelB) of Escherichia coli is a secondary transporter that couples the uptake of melibiose and various other galactosides to symport of cations that can be Na+, Li+ or H+. MelB belongs to the glycoside-pentoside-hexuronide: cation symporter family of porters and is suggested to have 12 transmembrane helices. We have determined the three-dimensional structure of MelB at 10A resolution in the membrane plane with cryo-electron microscopy from two-dimensional crystals. The three-dimensional map shows a heart-shaped molecule composed of two domains with a large central cavity between them. The structure is constricted at one side of the membrane while it is open to the other. The overall molecular shape resembles those of lactose permease and glycerol-3-phosphate transporter. However, organization of helices in MelB seems less symmetrical than in these two members of the major facilitator superfamily.     

The inner interhelix loop 4-5 of the melibiose permease from Escherichia coli takes part in conformational changes after sugar binding

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.     

Alteration of Sugar-Induced Conformational Changes of the Melibiose Permease by Mutating Arg in Loop 4-5

The melibiose permease (MelB) from Escherichia coli couples the uptake of melibiose to that of Na⁺, Li⁺, or H⁺. In this work, we applied attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy to obtain information about the structural changes involved in substrate interaction with the R141C mutant and with the wild-type MelB reacted with N-ethylmaleimide (NEM). These modified permeases have the ability to bind the substrates but fail to transport them. It is shown that the sugar-induced ATR-FTIR difference spectra of the R141C mutant are different from those corresponding to the Cys-less permease from which it is derived. There are alterations of peaks assigned to turns and β-structures located most likely in loop 4-5. In addition, and quite notably, a peak at 1659 cm⁻¹, assigned to changes at the level of one α-helix subpopulation, disappears in the melibiose-induced difference spectrum in the presence of Na⁺, suggesting a reduction of the conformational change capacity of the mutated MelB. These helices may involve structural components that couple the cation- and sugar-binding sites. On the other hand, MelB-NEM difference spectra are proportionally less disrupted than the R141C ones. Hence, the transport cycle of these two permeases, modified at two different loops, is most likely impaired at a different stage. It is proposed that the R141C mutant leads to the generation of a partially defective ternary complex that is unable to catalyze the subsequent conformational change necessary for substrate translocation.     

A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii

Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon. The melibiose transporter gene melB was cloned from a C. freundii mutant M4 that could utilize melibiose as a sole carbon source. Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation. Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter. The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport. The amino acid sequence of the C. freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB. These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.     

Selectivity of alkali cation influx across the plasma membrane of oat roots: cation specificity of the plasma membrane ATPase

Influx of alkali cations (Li(+), Na(+), K(+), Rb(+), Cs(+)) across plasma membranes of cells of excised roots of Avena sativa cv. Goodfield was selective, but different, in the absence and in the presence of 1 mm CaSO(4). Ca(2+) reduced the influx rates of all of the alkali cations-especially Na(+) and Li(+). Transport selectivity changed as the external concentrations of the alkali cations increased.Plasma membrane ATPase, purified from Avena sativa roots, was differentially stimulated by alkali cations. This specificity, however, was not altered by Ca(2+) or the external cation concentrations. A close correspondence existed between the relative influx rates of K(+), Rb(+), and Cs(+) and the relative stimulation of the ATPase by these cations. A similar correspondence did not occur for Na(+) and Li(+).Selective cation transport in oat roots could result, in part, from the specificity of the plasma membrane ATPase, but other factors such as specific carriers or porters or differential diffusion rates must also be involved.     

Facilitated diffusion properties of melibiose permease in Escherichia coli membrane vesicles. Release of co-substrates is rate limiting for permease cycling

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+). Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+. The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes. This suggests that the permease functions asymmetrically. Cross-comparison between the rates of net [3H]melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease. The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling. This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation. Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.     

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

Nucleotide-binding kinetics of Na,K-ATPase: cation dependence

Correlation between the Na,K-ATPase affinity to ADP and the cation (its nature and concentration) present in the medium was investigated. In buffer with low ionic strength (I approximately 1 mM) high-affinity ADP binding was not observed, while a stepwise increase in the concentrations of added cation (Na(+), Tris(+), imidazole(+), N-methylglucamine(+), choline(+)) induced an increase in the ADP affinity. The effect was fully saturated at 30-50 mM for all of the cations tested. The maximal affinity for ADP was slightly higher in the presence of Na(+), Tris(+), or imidazole(+) than in the presence of N-methylglucamine(+) or choline(+) (equilibrium dissociation constant K(d) 0.2-0.3 vs 0.7 microM). The ADP dissociation rates from its complex with enzyme in the presence of Na(+) or Tris(+) were similar, implying identity of the nucleotide-binding enzyme conformations, which therefore are assigned to E(1). The ability to compete with K(+) clearly distinguished Na(+) from other cations, which speaks against the sole involvement of the transport sites in the induction of the ADP-binding E(1) conformation. Since the cations are similar in their mode of induction of the high ADP affinity but they demonstrate a pronounced difference in ability to compete with K(+), their effects cannot be combined within any scheme with only one type of cation-binding sites. We suggest that the high affinity toward nucleotide is induced by cation interactions within the protein or lipid and that these nucleotide-domain-related sites coexist with the transport sites, which bind only Na(+) or K(+).     

Insights into the Inhibitory Mechanisms of the Regulatory Protein IIAGlc on Melibiose Permease Activity

The phosphotransfer protein IIA^Glc of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIA^Glc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA^Glc binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIA^Glc directly binds to MelB of Salmonella typhimurium (MelB_St) and Escherichia coli MelB (MelB_Ec) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3–10 μm for MelB_St and 25 μm for MelB_Ec. All of the binding is solely driven by favorable enthalpy forces. IIA^Glc binding to MelB_St in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIA^Glc-bound MelB_St exhibits a decreased binding affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIA^Glc inhibits the induced fit process and restricts the conformational dynamics of MelB_St.     

Extracellular pH modulates kinetics of the Na(+),K(+)-ATPase

To investigate effects of pH on the Na(+),K(+)-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K(+), and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na(+), K(+)-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na(+) ions with the pump molecule and/or conformational changes during Na(+)/Na(+) exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K(+) (< or =2 mM) and with 50 Na(+). This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na(+). External stimulation of the pump by K(+) in the absence of Na(+) can be described by a voltage-dependent K(m) value with an apparent valency z(K). At higher external pH the z(K) value is reduced. The transient current signal in the absence of external K(+) can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 micros and less than 100 micros during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about -120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K(+) and Na(+) binding.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

Residue Asp-189 controls both substrate binding and the monovalent cation specificity of thrombin

Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na(+). The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na(+)-activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin-like proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na(+) to the smaller cation Li(+) or to the larger cation Rb(+), respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na(+) (Asp-189) to Li(+) (Ser-189) or Rb(+) (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.