Structural basis of Na+ activation mimicry in murine thrombin

Unlike human thrombin, murine thrombin lacks Na+ activation due to the charge reversal substitution D222K in the Na+ binding loop. However, the enzyme is functionally stabilized in a Na+-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na+-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na+ binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na+ pore in the murine enzyme. These findings demonstrate that Na+ activation in thrombin is linked to the architecture of the Na+ pore. The molecular strategy of Na+ activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.     

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.     

Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.     

Crystal structure of the anticoagulant slow form of thrombin

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na(+)-free slow form. The slow form shows subtle differences compared with the currently available structures of the Na(+)-bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190-193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.     

Crystal Structure of human pyridoxal kinase: structural basis of M(+) and M(2+) activation

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5' alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K(+) when compared to Na(+); however, the maximal activity of the Na(+) form of the enzyme is more than double the activity in the presence of K(+). Other monovalent cations, Li(+), Cs(+), and Rb(+) do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 A resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg(2+) and Na(+) acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes.     

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.     

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.     

The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+). Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+). Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+). E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.     

Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

An extensive interaction interface between thrombin and factor V is required for factor V activation

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.     

Replacement of thrombin residue G184 with Lys or Arg fails to mimic Na+ binding

Na+ binding to thrombin enhances the catalytic activity toward numerous synthetic and natural substrates. The bound Na+ is located in a solvent channel 16 A away from the catalytic triad, and connects with D189 in the S1 site through an intervening water molecule. Molecular modeling indicates that the G184K substitution in thrombin positions the protonated epsilon-amino group of the Lys side-chain to replace the bound Na+. Likewise, the G184R substitution positions the guanidinium group of the longer Arg side-chain to replace both the bound Na+ and the connecting water molecule to D189. We explored whether the G184K or G184R substitution would replace the bound Na+ and yield a thrombin derivative stabilized in the highly active fast form. Both the G184K and G184R mutants lost sensitivity to monovalent cations, as expected, but their activity toward a chromogenic substrate was compromised up to 200-fold as a result of impaired diffusion into the S1 site and decreased deacylation rate. Interestingly, both G184K and G184R substitutions compromised cleavage of procoagulant substrates fibrinogen and PAR1 more than that of the anticoagulant substrate protein C. These findings demonstrate that Na+ binding to thrombin is difficult to mimic functionally with residue side-chains, in analogy with results from other systems.     

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.     

Crystal structure of thrombin in a self-inhibited conformation

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.     

Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.     

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.     

Potassium-selective block of barium permeation through single KcsA channels

Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.     

Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.     

Monovalent cation effects on the activity of the xenobiotic/medium-chain fatty acid:CoA ligases are substrate specific

The effect of monovalent cation on the activity of the XL-I and XL-III forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) was investigated using a variety of different carboxylic acid substrates. With benzoate or p-hydroxybenzoate as substrate, the XL-I ligase was essentially inactive in the absence of monovalent cation. However, with phenylacetic acid and medium-chain fatty acids as substrate, the enzyme retained 3 to 10% activity upon removal of monovalent cation. Further, while Na+ was ineffective with benzoate and p-hydroxybenzoate as substrates, it was effective with other substrates, although still less effective than K+. For XL-III, activity toward benzoate, hydroxybenzoate, and salicylate was insignificant in the absence of monovalent cation, but this rate was 10% of the K(+)-supported rate for hexanoate and 20% for decanoate. Also, with decanoate as substrate, XL-III was activated more by Na+ than by K+. Thus, the nature of the dependence on monovalent cation for activity is substrate-selective. Kinetic analysis of the effect of K+ on the activity of XL-I and XL-III revealed that activation by K+ was not the result of alteration of the affinity of the enzymes for either ATP or the carboxylic acid. For both forms of XM-ligase, K+ was found to enhance the affinity of the enzyme for CoA, regardless of the substrate, although the extent of the enhancement was substrate-specific. In almost all cases there was further activation, even at saturating concentrations of CoA, which indicates an additional effect of monovalent cation on the catalytic rate constant for the reaction. The exception was activation of XL-III activity toward decanoate, which was solely the result of enhanced binding affinity for CoA.     

Thrombin: a paradigm for enzymes allosterically activated by monovalent cations

Enzymes activated by monovalent cations are abundantly represented in plants and in the animal world. The mechanism, of activation involves formation of a ternary intermediate with the enzyme-substrate complex, or binding of the cation to an allosteric site in the protein. Thrombin is a Na+-activated enzyme with procoagulant, anticoagulant and signaling roles. The binding of Na+ influences allosterically thrombin function and offers a paradigm for regulatory control of protease activity and specificity. Here we review the molecular basis of thrombin allostery as recently emerged from mutagenesis and structural studies. The role of Na+ in blood coagulation and the evolution of serine proteases are also discussed.