Lignin peroxidase of Phanerochaete chrysosporium. Evidence for an acidic ionization controlling activity

The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.     

Chloride binding regulates the Schiff base pK in gecko P521 cone-type visual pigment

The binding of chloride is known to shift the absorption spectrum of most long-wavelength-absorbing cone-type visual pigments roughly 30 nm to the red. We determined that the chloride binding constant for this color shift in the gecko P521 visual pigment is 0.4 mM at pH 6.0. We found an additional effect of chloride on the P521 pigment: the apparent pKa of the Schiff base in P521 is greatly increased as the chloride concentration is increased. The apparent Schiff base pKa shifts from 8.4 for the chloride-free form to >10.4 for the chloride-bound form. We show that this shift is due to chloride binding to the pigment, not to the screening of the membrane surface charges by chloride ions. We also found that at high pH, the absorption maximum of the chloride-free pigment shifts from 495 to 475 nm. We suggest that the chloride-dependent shift of the apparent Schiff base pKa is due to the deprotonation of a residue in the chloride binding site with a pKa of ca. 8.5, roughly that of the Schiff base in the absence of chloride. The deprotonation of this site results in the formation of the 475 nm pigment and a 100-fold decrease in the pigment's ability to bind chloride. Increasing the concentration of chloride results in the stabilization of the protonated state of this residue in the chloride binding site and thus increased chloride binding with an accompanying increase in the Schiff base pK.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.     

Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide

Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.     

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).     

Kinetics of bicarbonate and chloride transport in human red cell membranes

Unidirectional [14C]HCO3- and 36Cl- efflux from human red cells and ghosts was studied under self-exchange conditions at pH 7.8 and 0 degrees C by means of the Millipore-Swinnex filtering technique. Control bicarbonate experiments showed that 14CO2 loss from the cells to the efflux medium was insignificant. The anion flux was determined under (a) symmetric variations of the anion concentration (C(i) = C(o) = 5-700 mM), and (b) asymmetric conditions with CAn constant on one side and varied on the other side of the membrane. Simple Michaelis-Menten-like kinetics (MM fit: J(eff) = J(eff)max.C/(K1/2 + C)) was used to describe anion flux dependence on C for (a) C(i) = C(o) = 5-100 mM, (b) C(i) = 6-100 mM, C(o) = constant, and (c) C(i) = constant, C(o) = 1-25 mM. At higher cellular concentrations noncompetitive self-inhibition by anion binding (inhibition constant Ki mM) to an intracellular site was included in the model (MS fit): J(eff) = J(eff)max.C(i)/[(K1/2 + C(i)).(1 + C(i)/Ki)]. The MM fits show that the external half-saturation constant, Ko1/2 ( = C(o)An for J(eff,o) = 1/2.j(eff,o)max) at C(o) = 1-25 mM is 1.5-2.4 mM (HCO3-) and 1.8-2.6 mM (Cl-). At C(o) = 1-260 mM Ko1/2 is 1.2-1.5 mM (HCO3-) and 1.4-1.8 mM (Cl-). The respective maximum flux, J(eff,o)max (nmol/[cm2.s]), for C(o) = 1-25 mM is 0.41-0.51 (HCO3-) and 0.28-0.38 (Cl-), and for C(o) = 1-260 mM 0.39-0.44 (HCO3-) and 0.27-0.31 (Cl-). The internal half-saturation constant, Ki1/2 mM is: MM fit (C(i) = 6-100 mM, C(o) = 50 mM), 18.0 mM (HCO3-) and 23.8 mM (Cl-); MS fit (C(i) = 6-920 mM, C(o) = 50 mM), 32.0 mM (HCO3-) and 45.1 mM (Cl-). The maximum flux, J(eff,i)max (nmol/[cm2.s]) is: MM fit; 0.50 (HCO3-) and 0.34 (Cl-); MS fit, 0.70 (HCO-3) and 0.50 (Cl-). The half-inhibition constants of the MS fit, Ki, are 393 mM (HCO3-) and 544 mM (Cl-). The MM fit shows that the symmetric half-saturation constant, Ks1/2, is 20.2 (HCO-3) and 23.9 (Cl-) mM, and J(eff,s)max is 0.51 (HCO3-) and 0.32 (Cl-) nmol/(cm2.s). The MS fit shows that for C = 5-700 mM Ks1/2 is 30.4 nM (HCO3-) and 50.1 mM (Cl-), and Ki is 541 mM (HCO3-) and 392 mM (Cl-).(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of anions and pH on the conformational change of horse liver alcohol dehydrogenase induced by binding of oxidized nicotinamide adenine dinucleotide: binding of chloride to the catalytic metal ion

The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.     

Evidence from electron paramagnetic resonance for function-related conformation changes in the anion-transport protein of human erythrocytes

The erythrocyte membrane protein involved in anion transport (band 3) was isolated in its native lipid milieu in the form of leaky vesicles and then was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-meleimide (MalMe4PipO). The resulting electron paramagnetic resonance spectrum of band-3-bound MalMe4PipO was resolved into a rapid tumbling component and another, relatively immobile component. The percentage of the signal contributed by the mobile component (Q), was sensitive to various characteristic factors known to affect erythrocyte anion transport: Q was a hyperbolic function of chloride concentration displaying a half-saturation constant K1/2 similar to that of chloride transport. On the other hand Q showed a biphasic response to sulfate concentration, in line with the relatively high affinity of sulfate for the anion modifier site. Q was a saturable function of pH, either in presence of Cl- or SO4(-2), showing a pKa between pH 6.0 and 6.5, in analogy with the pH titration curve of Cl- and SO4(-2), transport. Spin-labelled vesicles treated with a covalent inhibitor of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, were markedly less susceptible to changes in Cl- concentration. It is suggested that the electron paramagnetic resonance spectrum of MalMe4PipO covalently bound to the band-3 protein, reports conformational changes which are related to the anion-transport function of this protein.     

Activation of angiotensin converting enzyme by monovalent anions

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.     

Effects of two ionizing groups on the active site of human carbonic anhydrase B.

Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B.     

Kinetic analysis by stopped-flow radiationless energy transfer studies: effect of anions on the activity of carboxypeptidase A

We have utilized a highly sensitive radiationless energy transfer (RET) assay to investigate the effect of anions on the activity of carboxypeptidase A (CPD-A). The RET kinetic method visualizes the ES complex directly and thus enables both the mode of action of anions and the quantitation of their effect to be determined at a single substrate concentration. In marked contrast to the activating effect of anions on the closely related metalloprotease, angiotensin converting enzyme, Cl-, and other anions inhibit CPD-A catalysis. NaCl inhibits the hydrolysis of Dns-Ala-Ala-Phe throughout the pH range 6-10. Other di- and tripeptides are similarly inhibited while their ester analogues are affected only slightly. Changes in the type of cation [e.g., Na+, Li+, K+, Ca2+, and (CH3)4N+] at a constant [Cl-1] of 0.1 M showed no difference in the extent of inhibition, whereas with anion substitution the differences were marked. In all cases, the inhibition was partially competitive. At pH 5.9, the Ki values for the free enzyme are 51 (Cl-), 17 (N3-), 2.1 (SO4(2-)), and 0.21 mM (H2PO4-), and for the ES complex, the KI' values are 1000, 720, 42, and 13 mM, respectively. The other anions were shown to act at the chloride site. The results indicate that investigations of anion inhibition in 1 M NaCl, a typical assay condition, may be greatly hindered by the presence of Cl-. Thus, the competitive binding mode of phenylacetate toward peptide hydrolysis is greatly decreased by the presence of 1 M Cl- ion while its noncompetitive component is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical demonstration of HCO3--und Cl--dependent ATPase activities in the rectum of larval dragonflies and inhibition of rectal chloride uptake by thiocyanate (author's transl)

Hydrogencarbonate and chloride activated, ouabain-insensitive ATPase activities are demonstrated in the salt-absorbing rectum of larval dragonflies. Maximal activation is achieved at approx. 30 mM HCO3- and 20 mM Cl-, respectively. The stimulation of each anion obeys Michaelis-Menten kinetics Km values are 4.65 mM for HCO3-- and 10.25 mM for Cl--activation. The activating anion of one type of ATPase simultaneously exerts an inhibitory effect on the other. Cl--activation is also reduced by Mg.ATP in concentrations above 0.5 mM and by Tris-Hepes buffer exceeding 2.5 mM. Both anion-dependent ATPase activities are found enriched in subcellular membraneous fractions of the rectum. Thiocyanate inhibits both activities and causes a significant decrease in rectal uptake of radioactive chloride from hypo-osmotic external solution. In the case of HCO3- dependent ATPase a competitive inhibition as SCN- was found with an inhibitor constant of Ki=0.5 mM.     

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.     

Evidence for an essential histidine in neutral endopeptidase 24.11

Rat kidney neutral endopeptidase 24.11, "enkephalinase", was rapidly inactivated by diethyl pyrocarbonate under mildly acidic conditions. The pH dependence of inactivation revealed the modification of an essential residue with a pKa of 6.1. The reaction of the unprotonated group with diethyl pyrocarbonate exhibited a second-order rate constant of 11.6 M-1 s-1 and was accompanied by an increase in absorbance at 240 nm. Treatment of the inactivated enzyme with 50 mM hydroxylamine completely restored enzyme activity. These findings indicate histidine modification by diethyl pyrocarbonate. Comparison of the rate of inactivation with the increase in absorbance at 240 nm revealed a single histidine residue essential for catalysis. The presence of this histidine at the active site was indicated by (a) the protection of enzyme from inactivation provided by substrate and (b) the protection by the specific inhibitor phosphoramidon of one histidine residue from modification as determined spectrally. The dependence of the kinetic parameter Vmax/Km upon pH revealed two essential residues with pKa values of 5.9 and 7.3. It is proposed that the residue having a kinetic pKa of 5.9 is the histidine modified by diethyl pyrocarbonate and that this residue participates in general acid/base catalysis during substrate hydrolysis by neutral endopeptidase 24.11.     

Activating anions that replace Cl- in the O2-evolving complex of photosystem II slow the kinetics of the terminal step in water oxidation and destabilize the S2 and S3 states

Photosystem II, the multisubunit protein complex that oxidizes water to O2, requires the inorganic cofactors Ca2+ and Cl- to exhibit optimal activity. Chloride can be replaced functionally by a small number of anionic cofactors (Br-, NO3-, NO2-, I-), but among these anions, only Br- is capable of restoring rates of oxygen evolution comparable to those observed with Cl-. UV absorption difference spectroscopy was utilized in the experiments described here as a probe to monitor donor side reactions in photosystem II in the presence of Cl- or surrogate anions. The rate of the final step of the water oxidation cycle was found to depend on the activating anion bound at the Cl- site, but the kinetics of this step did not limit the light-saturated rate of oxygen evolution. Instead, the lower oxygen evolution rates supported by surrogate anions appeared to be correlated with an instability of the higher oxidation states of the oxygen-evolving complex that was induced by addition of these anions. Reduction of these states takes place not only with I- but also with NO2- and to a lesser extent even with NO3- and Br- and is not related to the ability of these anions to bind at the Cl- binding site. Rather, it appears that these anions can attack higher oxidation states of the oxygen evolving complex from a second site that is not shielded by the extrinsic 17 and 23 kDa polypeptides and cause a one-electron reduction. The decrease of the oxygen evolution rate may result from accumulated damage to the reaction center protein by the one-electron oxidation product of the anion.     

Purification and some characteristics of a non-haem bromoperoxidase from Streptomyces aureofaciens

A bromoperoxidase was isolated from the chlortetracycline-producing actinomycete, Streptomyces aureofaciens. This enzyme catalysed bromination and iodination, but surprisingly did not catalyse chlorination. The enzyme had an acidic pH optimum (pH 4.3) and the isoelectric point was 3.5. The Km for bromide was 20 mM and the Km for H2O2 was as high as 8 mM. The bromoperoxidase did not contain haem, since it was not inhibited by azide or cyanide. Excess bromide or chloride had no effect on its brominating activity; however, fluoride strongly inhibited the bromoperoxidase (Ki = 20 microM). On the basis of gel electrophoresis in the absence and presence of sodium dodecyl sulphate, the molecular mass of the enzyme was 65 kDa and it consisted of two subunits of 32 kDa each. The bromoperoxidase was remarkably thermostable.     

Kinetics of reaction of anions with methemerythrin derivatives.

The kinetics of anation of methemerythrin over a wide range of pH and concentration of anions have been studied at 25 degrees C. The azide and thiocyanate ions have been most intensively investigated but experiments with fluoride and chloride are also reported. The replacement of anion in methemerythrin-anionic adducts by other anions has also been studied. Except for replacement of met-fluoride by azide, all replacements can be explained by a dissociative mechanism via the aquated species. Anations are second-order and an associative mechanism is preferred. The second-order rate constant decreases with increasing anion concentrations (from 20 muM to 20 mM). This is attributed to the effect of a secondary anion binding site. The behavior of octameric and monomeric forms of the protein toward thiocyanate is identical. A comparison of results with simple Fe(III) complexes and certain metalloproteins is made.     

Binding of Escherichia coli ribosomal protein S8 to 16S rRNA: kinetic and thermodynamic characterization

A sensitive membrane filter assay has been used to examine the kinetic and equilibrium properties of the interactions between Escherichia coli ribosomal protein S8 and 16S rRNA. In standard conditions (0 degrees C, pH 7.5, 20 mM Mg2+, 0.35 M KCl) the apparent association constant is 5 +/- 0.5 X 10(-7) M-1. The interaction is highly specific, and the kinetics of the reaction are consistent with the apparent association constant. Nevertheless, the rate of association is somewhat slower than that expected for a diffusion-controlled reaction, suggesting some steric constraint. The association is only slightly affected by temperature (delta H = -1.8 kcal/mol). The entropy change [delta S = +29 cal/(mol K)] is clearly the main driving force for the reaction. The salt dependence of Ka reveals that five ions are released upon binding at pH 7.5 and in the presence of 10 mM magnesium. The substitution of various anions for Cl- has an appreciable effect on the magnitude of Ka, following the order CH3COO- greater than Cl- greater than Br-, thus indicating the existence of anion binding site(s) on S8. An equal number of ions were released when Cl- was replaced by CH3COO-, but the absence of anion release upon binding cannot be excluded. On the other hand, the free energy of binding appears not to be exclusively electrostatic in nature. The effect of pH on both temperature and ionic strength dependence of Ka has been examined. It appears that protonation of residue(s) (with pK congruent to 9) increases the affinity via a generalized charge effect. On the other hand, deprotonation of some residue(s) with a pK congruent to 5-6 seems to be required for binding. Furthermore, the unique cysteine present in S8 was shown to be essential for binding.     

Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6- fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.     

The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate [PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.