Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase

Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C. The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present. When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.     

Essential arginine residues in tryptophanase from Escherichia coli.

Tryptophanase from Escherichia coli B/1t7-A is inactivated by the arginine-specific reagent, phenylglyoxal, in potassium phosphate buffer at pH 7.8 AND 25 degrees. Apo- and holoenzyme are inactivated at the same rate, and inactivation of both is correlated with modification of 2 arginine residues/tryptophanase monomer. Substrate analogs having a carboxyl group protect the holoenzyme against both inactivation and arginine modification but have no effect on the inactivation or modification of the apoenzyme. Phenylglyoxal-modified apotryptophanase retains the capacity to bind the coenzyme, pyridoxal-P, but the spectrum of this reconstituted species differs from that of native holotryptophanase. Neither this reconstituted species nor the phenyglyoxal-modified holoenzyme shows the 500 nm absorption characteristic of the native enzyme when substrates are added. These results demonstrate a requirement for specific arginine residues for substrate binding and are discussed in the context of the known conformational and spectal forms of tryptophanase with regard to a possible role for arginine residues in formation of a catalytically effective enzyme-pyridoxal-P complex.     

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.     

Effect of monovalent cations on the catalytic and spectral properties of tyrosine-phenol-lyase from Citrobacter intermedius

The action of monovalent cations Li+, Na+, K+, Rb+, Cs+, NH4+ on catalytic and physico-chemical properties of bacterial tyrosine--phenol-lyase was investigated. It was shown that K+, Rb+, Cs+, NH4+ were the noncompetitive activators of the enzyme, Na+ was an inhibitor, Li+ did not influence the catalytic activity. The values of KA and Vmax were determined for the activators in the reaction of alpha, beta-elimination of L-tyrosine. Monovalent cations affect the absorption and CD spectra of the enzyme and its complex with the quasi-substrate--L-alanine. It was suggested that an activation of tyrosine phenollyase by monovalent cations was connected with the increase of the active protonated form of the holoenzyme (lambda max 420 mm) induced by the cations-activators.     

Interaction of valinomycin and monovalent cations with the (Ca2+,Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum

The interactions of monovalent cations and of the K+-specific ionophore, valinomycin, with the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum have been studied in the absence of cation gradients by their effects on enzyme turnover and on the ATP plus Ca2+-dependent enhanced fluorescence of the ATP analogue, 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine 5'-triphosphate (TNP-ATP) (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Monovalent cations decreased turnover-dependent TNP-ATP fluorescence in the series K+ greater than Rb+ approximately equal to Cs+ greater than Na+ greater than Li+ (K0.5 = 49, 73, 75, 94, and 246 mM, respectively), consistent with the known specificity of the monovalent cation binding site that stimulates turnover and E-P hydrolysis. Valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased ATPase activity by 30% and abolished the stimulatory effects of 150 mM KCl or NaCl on turnover. The ionophore alone enhanced TNP-ATP fluorescence by 20% and altered the specificity and affinity of the site that inhibited TNP-ATP fluorescence to Cs+ greater than Rb+ greater than K+ approximately equal to Na+ greater than Li+ (K0.5 = 79, 111, 134, 136, and 270 mM, respectively), which follows the Hofmeister series for effectiveness of monovalent lyotropic cations. TNP-ATP binding was not affected by either monovalent cations or valinomycin. Inhibition of turnover-dependent TNP-ATP fluorescence appears to be a useful parameter for monitoring monovalent cation binding to the Ca2+-ATPase. It is concluded that the ionophore interacts directly with the Ca2+-ATPase, independent of its K+ conductance effects on the lipid bilayer, and modifies the affinity and specificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex.     

The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.     

Elucidation of a monovalent cation dependence and characterization of the divalent cation binding site of the fosfomycin resistance protein (FosA).

The fosfomycin resistance protein FosA is a member of a distinct superfamily of metalloenzymes containing glyoxalase I, extradiol dioxygenases, and methylmalonyl-CoA epimerase. The dimeric enzyme, with the aid of a single mononuclear Mn2+ site in each subunit, catalyzes the addition of glutathione (GSH) to the oxirane ring of the antibiotic, rendering it inactive. Sequence alignments suggest that the metal binding site of FosA is composed of three residues: H7, H67, and E113. The single mutants H7A, H67A, and E113A as well as the more conservative mutants H7Q, H67Q, and E113Q exhibit marked decreases in the ability to bind Mn2+ and, in most instances, decreases in catalytic efficiency and the ability to confer resistance to the antibiotic. The enzyme also requires the monovalent cation K+ for optimal activity. The K+ ion activates the enzyme 100-fold with an activation constant of 6 mM, well below the physiologic concentration of K+ in E. coli. K+ can be replaced by other monovalent cations of similar ionic radii. Several lines of evidence suggest that the K+ ion interacts directly with the active site. Interaction of the enzyme with K+ is found to be dependent on the presence of the substrate fosfomycin. Moreover, the E113Q mutant exhibits a kcat which is 40% that of wild-type in the absence of K+. This mutant is not activated by monovalent cations. The behavior of the E113Q mutant is consistent with the proposition that the K+ ion helps balance the charge at the metal center, further lowering the activation barrier for addition of the anionic nucleophile. The fully activated, native enzyme provides a rate acceleration of >10(15) with respect to the spontaneous addition of GSH to the oxirane.     

The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per beta monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca2+ or spermidine(3+) but not with the monovalent cations Na+ and K+. The Mg2+-induced fluorescence enhancement was specific for FM-beta and was not observed with FM-glutathione, dimethoxystilbenemaleimide-beta, or pyrenylmaleimide-beta. Gel filtration studies indicated that the beta dimer-monomer dissociation occurred at physiologically significant beta concentrations and that the presence of 10 mM Mg2+ shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies beta dissociation is due to a relief from homoquenching of FM when the beta dimer dissociates into monomers.     

Effects of monovalent cations on AMP nucleosidase from Azotobacter vinelandii.

The effect of monovalent cations on the purified AMP nucleosidase (AMP phosphoribohydrolase, EC 3.2.2.4) from Azotobacter vinelandii was investigated. All the monovalent cations were activators of the enzyme: Rb+ and Cs+ were the most effective, followed by K+, Na+, NH4+ and Li+ in that order. The apparent Ka for MgATP and nH values (Hill's interaction coefficient) decreased from 0.9 to 0.1 mM, and from 4 to 1, respectively, with the increase in K+ concentration, suggesting that the cation effects are on MgATP binding rather than catalysis. Gel filtration studies have revealed that the enzyme forms a non-dissociable enzyme species with a Stokes radius of 6.0--6.2 nm in the presence of saturating concentrations of monovalent cations, which can be distinguished from the 5.5-nm enzyme species showing temperature-dependent dissociation of the molecule in sulfate or phosphate. These results suggest that these ligands affect the association of the subunits through changes in the environment of the hydrophobic side chains of the enzyme molecules.     

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.     

Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli

We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E. coli K-12 MD55 with a vector plasmid, pBR322. The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein. With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method. These single crystals appeared to be suitable for X-ray diffraction analysis.     

Monovalent cation activation of tryptophanase.

The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods. Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3). It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton. On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E. (1967) J. Biol. Chem; 242, 2800-2809. The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active. When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+. Two moles of thallium (I) were bound per mole of subunit. The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.     

Principles of selectivity of sodium and potassium binding sites of the Na+/K+-ATPase. A corollary hypothesis

The mechanisms whereby the sodium and potassium binding sites of heart sacrolemmal Na+/K+-ATPase (EC 3.6.1.3) distinguished between monovalent cations were investigated using methods of enzyme kinetics. The properties of the sodium binding sites were studied in the presence of 2,4,6-trinitrobenzenesulfonic acid in concentrations completely inhibiting the action of potassium on the enzyme. To test the selectivity of potassium binding sites, K+-p-nitrophenylphosphatase activity was employed as a model. The results suggest that the selectivity of Na+- and K+-binding sites of Na+/K+-ATPase may be due to two independent mechanisms: (i) The principle of key and lock (formation of coordination bounds); (ii) Optimal difference between solvatation energy (in the specific binding site) and hydration enthalpy of the respective cation.     

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+.     

Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.     

Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.     

Heart sarcolemmal (Na+ + K+)-ATPase has an essential amino group in the potassium binding site on the enzyme molecule

Selective modification of primary amino groups of (Na+ + K+)-ATPase by trinitrobenzene sulfonic acid (TNBS) resulted in a considerable inhibition of the specific activity of the enzyme. Investigation by means of enzyme and sorption kinetics of activation of heart sarcolemmal (Na+ + K+)-ATPase by its monovalent cationic ligands added simultaneously with TNBS revealed: a considerable competition between K+-ions and TNBS for the potassium binding site on the enzyme molecule; a non-competitive type of inhibition of Na+-induced activation of the enzyme. Both, potassium and sodium ions depressed, and magnesium ions enhanced the initial rate of TNBS-sorption; however, none of the above cations influenced the equilibrium value of TNBS sorption onto isolated sarcolemmal membranes. Ouabain, on the other hand, did not inhibit the initial rate and decreased the equilibrium value of TNBS sorption onto the membranes. The results obtained enabled the identification of an essential amino group in the potassium binding site of the (Na+ + K+)-ATPase molecule.     

Monovalent cation binding to cubic insulin crystals.

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.     

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

Channel permeant cations compete selectively with noncompetitive inhibitors of the nicotinic acetylcholine receptor.

Previous work suggests that noncompetitive inhibitor (NCI) ligands and channel permeant cations bind to sites within the nicotinic acetylcholine receptor ion channel. We have used ethidium as a fluorescent probe of the NCI site to investigate interactions between NCI ligands and channel permeant cations. We found that ethidium can be completely displaced from the receptor by a variety of inorganic monovalent and divalent cations. The rank order of monovalent cation affinities was found to be Tl+ greater than Rb+ greater than or equal to K+ greater than Cs+ greater than Na+ greater than Li+. The monovalent cation Kd values vary markedly over a 40-fold range, from 3 to 121 mM. The Kd values and rank order correspond to values determined previously from electrophysiological data. Hill plots of the back titrations yield slopes of 1.0 for all monovalent cations, indicating a single class of independent sites, as shown previously for NCI ligands. Scatchard analysis of ethidium binding in the presence of Tl+ reveals a reduction in affinity and no changes in the maximal number of sites. In the presence of agonist the kinetics of ethidium dissociation induced by the addition of phencyclidine or cations alone or the simultaneous addition of both are nearly identical. The ethidium dissociation rate induced by either phencyclidine or cations is regulated by the occupation of the agonist sites in a similar manner. These results indicate that the effect of cations on NCI ligand binding occurs by mutually exclusive competition. We suggest that NCIs can regulate cation binding at a physiological cation recognition site that is likely part of the cation permeation path through the receptor channel.