An essential role of active site arginine residue in iodide binding and histidine residue in electron transfer for iodide oxidation by horseradish peroxidase

The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controlling iodide oxidation using chemical modification technique. The arginine specific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 25.12 min^-1 M^-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can be protected both by iodide and an aromatic donor such as guaiacol. Moreover, guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection constant (K_p) for iodide and guaiacol are 500 and 10 M respectively indicating higher affinity of guaiacol than iodide at this site. Donor binding studies indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased K_d value to 571 mM from the native enzyme (K_d = 150 mM). Although arginine-modified enzyme reacts with H₂O₂ to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding.The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order rate constant of 0.66 min^-1 M^-1. Both the nitrogens (, ) of histidine imidazole were modified as evidenced by the characteristic peak at 222 nm. The enzyme is not protected by iodide suggesting that imidazolium ion is not involved in iodide binding. Moreover, DEPC-modified enzyme binds iodide similar to the native enzyme. However, the modified enzyme does not form compound II but forms compound I only with higher concentration of H₂O₂ suggesting the catalytic role of this histidine in the formation and autoreduction of compound I. Interestingly, compound I thus formed is not reduced by iodide indicating block of electron transport from the donor to the compound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the heme ferryl group during oxidation.     

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [¹⁴C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the ¹⁴C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.     

The effect of neutral salt anions on the oxidative deamination activity of plant glutamate dehydrogenase

Increasing concentrations of anions of the Hofmeister series decrease the activity of highly purified glutamate dehydrogenase (EC 1.4.1.2.) from Pisum sativum L. The extent of the inactivation, as estimated by the ion concentration which causes a 50% transformation of the native form to the low activity form of the enzyme (approximately halfmaximal activity), follows the ranking Cl^–––3 ^– –. Sulfate has a slightly activating effect. At salt concentrations higher than 1 M (with SCN^– higher than 200 mM), the activity decreases to a value from 3–6% of the initial activity and remains then stable over a wide range of higher anion concentrations. From kinetic investigations it is seen that the treatment of the enzyme with anions decreases the affinity for the cosubstrate NAD⁺ and the substrate L-glutamate (K _M-values increased) and also increases the dissociation constant for NAD⁺. The salt induced inactivation is reversible by dilution. From a mathematical treatment of the kinetic data of the inactivation, it is seen that increasing concentrations of the anions exert cooperative effects on the inactivation process.     

Chemical modifications of histidine residues in cytoplasmic asparate aminotransferase from beef kidney

Summary Holo and apoenzyme of aspartate aminotransferase from beef kidney are 80% inactivated by photoxidation in the presence of 2 × 10^–6 m tetraiodofluroescein with the modification of two histidine residues per enzyme protomer. At a higher concentration (1 × 10^–5 m) a tyrosine residue is also modified. The keto substrates, ketoglutarate and oxalacetate, protect the enzyme from photoxidation.Diethylpyrocarbonate modifies three histidine residues per enzyme protomer and reduces the activity only 10%. These results suggest that the two histidine residues photoxidized through the sensitizer, are located in the active site of the enzyme, at least one of these appears to be involved in ketosubstrate binding. The other three histidines modified by diethylpyrocarbonate are likely located on the enzyme surface and are not involved in the catalytic activity of the enzyme.This work is part of a program supported by a grant from the Consiglio Nazionale delle Ricerche.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

Evidence for a functionally important histidine residue in human tyrosine hydroxylase

Summary Recombinant human tyrosine hydroxylase isozyme 1 (hTH1) shows a time- and concentration-dependent loss of catalytic activity when incubated with diethylpyrocarbonate (DEP) after reconstitution with Fe(II). The inactivation follows pseudo-first order kinetics with a second order rate constant of 300 M^–1 min^–1 at pH 6.8 and 20°C and is partially reversed by hydroxylamine. The difference absorption spectrum of the DEP-modified vs native enzyme shows a peak at 244 nm, characteristic of mono-N-carbethoxy-histidine. Up to five histidine residues are modified per enzyme subunit by a five-fold excess of the reagent, and two of them are protected from inactivation by the active site inhibitor dopamine. However, derivatization of only one residue appears to be responsible for the inactivation. Thus, no inactivation by DEP was found when the apoenzyme was preincubated with this reagent prior to its reconstitution with Fe(II), modifying four histidine residues.Abbreviations BH₄ (6R)-l-erythro-tetrahydrobiopterin - DEP diethylpyrocarbonate - DOPA 3,4-dihydroxyphenylalanine - hTH1 human tyrosine hydroxylase isoenzyme 1 - apo-hTH1 apoenzyme of hTH1 - Fe(II)-hTH1 holoenzyme (iron reconstituted) of hTH1 - dopamine-Fe(III)-hTH1 holoenzyme of hTH1 with dopamine bound - TH tyrosine hydroxylase     

Presence of an essential arginyl residue in D-β-hydroxybutyrate dehydrogenase from mitochondrial inner membrane

D-β-hydroxybutyrate dehydrogenase, a lipid requiring enzyme, is rapidly and completely inactivated by phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione. Inactivation, which occurs at the millimolar range, depends on the nature of buffer, borate ions are required to get enzyme inactivation by 2,3-butanedione. Most of the inactivation follows a pseudo first order kinetics, the stoichiometry being of one to one. Presence of NAD⁺ or methylmalonate (a substrate-like compound) prior addition of inhibitor does not affect inactivation, while methylmalonate in presence of NAD⁺ strongly protects against inactivation. Chemical modification of the enzyme does not affect K_D of NAD while K_M of β-hydroxybutyrate and Ki of methylmalonate (protecting agent) increase. In view of the high specificity of these inhibitors for arginyl residues of proteins, these results are in favour of the presence of at least one arginyl residue essential for enzyme activity and located in, or near the substrate binding site.     

Chicken liver fatty acid synthetase dimer: Evidence of asymmetrical structure from iodoacetamide inhibition studies

The condensing component of chicken liver fatty acid synthetase is inhibited by a sulfhydryl reagent, iodoacetamide, with a second-order rate constant of 0.23 M^–1 sec^–1 at pH 7.0 and 0. Complete inactivation requires the modification of approximately 8-SH groups per dimer of the enzyme. Quantitation of the extent of inactivation in the presence of i mM acetyl CoA (which completely protects the enzyme against inactivation) and in its absence shows that complete inactivation results from the binding of approximately 1.1 tool of carboxamidomethyl residues per dimer. These data are consistent with the proposed functional asymmetry of the enzyme.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M^–1 min^–1. The pH–inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.     

Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Summary The utility of the lipophilic anion thiocyanate (SCN⁺) as a probe for the indirect estimation of the cell membrane potential (V _m ) in Ehrlich ascites tumor cells has been evaluated by comparison to direct electrophysiological measurements. SCN accumulation is consisten with first-order uptake into a single kinetically-identifiable cellular compartement, achieving steadystate distribution in 20–30 min at 22°C. The steady-state distribution ratio ([SCN^–] _c /[SCN^–] _e ) in physiological saline is 0.44±0.02. Treatment of the cells with proparanolol (0.13 mM), an activator of Ca²⁺ dependent K⁺ channels, reduces the steady-state distribution ratio to 0.19±0.02. Conversely, treatmetn with BACl₂ (10 mM), an antagonist of the pathway, increases the SCN^– distribution ratio to 0.62±0.01. The equilibrium potentials (V _SCN ) calculated under these conditions are virtually identical to direct electrophysiological measurements of theV _m made under the same conditions. The effect of varing extracellular [K⁺]([K⁺] _e ) in the presence of constant [Na⁺] _e =100 mM has also been tested. In control cells, elevation of [K⁺] _e from 6 to 60 mM reducesV _SCN from –20.6±1.0 to –13.2±1.2 mV. Again, microelectrode measurements give excellent quantitative agreement. Propranolol increases the sensitivity of the cells to varying [K⁺] _e , so that a 10-fold elevation reducesV _SCN by approximately 31 mV. BaCl₂ greatly reduces this reponse: a 10-fold elevation in [K⁺] _e yielding only a 4-mV rediction inV _SCN . It is concluded that the membrane potential of Ehrlich cells can be estimated accurately from SCN^– distribution measurements.     

Calcium-Binding Properties of the Mitochondrial Channel-Forming Hydrophobic Component

A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa²⁺-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca²⁺ with a K_d of 8 × 10^–6 M, while at pH7.5 this K_d was decreased to 9 × 10^–5 M. B_max for the Ca²⁺-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca²⁺/g component were boundor one calcium ion to eight molecules of the component. The Ca²⁺ binding was stronglydecreased by 50–100 mM Na⁺, but not by K⁺. Treatment of mitochondria withCaCl₂ priorto ethanolic extraction resulted in a high level of Ca²⁺-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca²⁺-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.     

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 µM) and one with lower affinity (K_d = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 µM). The binding of [³⁵S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 µM) and six at the liver enzyme (K_d = 12 µM). The Paracoccus enzyme had only one binding site (K_d = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)     

Affinity labeling of oxaloacetate decarboxylase by novel dichlorotriazine linked alpha-ketoacids

The 4-aminophenyloxanilic acid and -mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (APOD) or -mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25°C shows a time-dependent and concentration-dependent loss of enzyme activity. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactivation following the Kitz & Wilson kinetics for time-dependent irreversible inhibition. The observed rate of enzyme inactivation (k _obs) exhibits a non-linear dependence on APOD or MPD concentration with maximum rate of inactivation (k ₃) of 0.013 min^–1 and 0.0046 min^–1 and K _D equal to 20.3 and 156 M respectively. The inactivation of oxaloacetate decarboxylase by APOD and MPD is competitively inhibited by OXAD substrate and inhibitors, such as oxaloacetate, ADP and oxalic acid whereas Mn⁺² enhances the rate of inactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatization by the label. These results show that APOD and MPD demonstrate the characteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Chemical modification of the α-amylase ofBacillus caldovelox with diethyl pyrocarbonate: Evidence for an essential histidine at the active site

Summary The -amylase ofBacillus caldovelox is inactivated by diethyl pyrocarbonate at pH 6.6 and 20°C by a monomolecular reaction with a second-order rate constant of 41.7 M^–1·min^–1. The rate of inactivation increases with decreasing pH, suggesting participation of an amino acid residue with a pK a of 6.6. The increase in absorbance at 240 nm, unchanged absorbance at 280 nm and reactivation in the presence of hydroxylamine suggest the participation of a histidine residue. Statistical analyses of inactivation suggest that only one histidine residue is essential for activity. Substrate afforded complete protection against inactivation, indicating the involvement of the histidine residue at the active site of the enzyme.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of35S thiocyanate

Summary Up to a SCN^– concentration of about 110mm, the concentration dependence of SCN^– equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits arans acceleration by Cl^– and can be inhibited by H₂DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced intrans acceleration experiments with Cl^– and insensitive to H₂DIDS but susceptible to inhibition by phloretin. At SCN^– concentrations exceeding 110mm, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered.The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0mm at pH 7.4 to 13.3mm at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19×10^–12 to 22×10^–12 moles×cm^–2×sec^–1. The nonsaturable flux component also increases slightly.In accordance with previous observations of Wieth (J. Physiol. (London) 207:563–580, 1970), we find that SCN^– increases K⁺ and Na⁺ permeability. The induced cation-permeability is considerably smaller than the SCN^– exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.     

The amyloidogenic region of the human prion protein contains a high affinity (Met)2(His)2 Cu(I) binding site

The prion protein (PrP^c) is a cuproprotein implicated in a number of human neurodegenerative diseases. Although many physiological functions have been ascribed to PrP, its potential to act as a neuronal antioxidant, based in part on its copper binding ability, is controversial and unresolved. A number of studies have shown that copper bound to PrP^c is not redox silent, and recent data shows that the Cu(II) sites at histidines 96 and 111 display reversible electrochemistry. Reversible electrochemistry implies redox cycling whilst the metal remains bound and with the absence of permanent oxidation or reduction of the protein. Despite this indirect evidence of Cu(I) binding to PrP, the nature of the Cu(I) binding site/s is unclear, although previous extended X-ray absorption fine structure (EXAFS) data has implicated methionines in the Cu(I) binding site. Using spectroscopic techniques we find that the PrP region encompassing histidines 96 and 111 can bind a Cu(I) ion in a site comprising His 96, His 111, Met 109 and Met 112. The four-coordinate (His)₂(Met)₂ Cu(I) site has a K_d = 10⁻¹⁵–10⁻¹² M indicative of high affinity. Mutation of histidine residues reduces the Cu(I) affinity. Although alluding to the fact the PrP could act in a direct superoxide dismutase-like fashion, the Cu(I)–PrP(91–124) site and affinity is comparable to that observed for bacterial periplasmic Cu(I) transporters.