The effect of chloride on the redox and EPR properties of myeloperoxidase

Myeloperoxidase was purified from human polymorphonuclear leukocytes and the effect of chloride upon the EPR and potentiometric properties was studied. The redox titration between the ferrous and ferric states of the enzyme yielded n = 1 Nernst plots between pH 9 and 4, with clear isosbestic points in the optical spectra during the redox change. The midpoint potential (Em) between the ferric and ferrous forms of the enzyme exhibited a pH-dependent change between pH 4 and 9, and the effect of added chloride ion indicated that Cl- competed with OH- for a binding site on the enzyme. Interestingly, the pH dependence of the Em indicated that the overall redox reactions of the enzyme was: ferric myeloperoxidase + 2e- + 1H+ = ferrous myeloperoxidase. Myeloperoxidase exhibited a rhombic high spin EPR signal which exhibited reduced rhombicity upon the binding of chloride. Our results strongly suggest that chloride binds to the sixth coordination position of the chlorin iron in myeloperoxidase by replacing the water which is the sixth ligand in the resting state. It is also concluded that the two iron centers are identical and that there is no interaction between them.     

Spectroscopic, ligand binding, and enzymatic properties of the spleen green hemeprotein. A comparison with myeloperoxidase

The bovine spleen green hemeprotein, a peroxidase which exhibits spectrophotometric properties similar to those of granulocyte myeloperoxidase, was purified using an improved method. The ligand affinity of the ferric enzyme was spectroscopically determined using chloride and cyanide as exogenous ligands. The pH dependence of the apparent dissociation constant of the enzyme-chloride complex showed the presence of a proton dissociable group with a pKa value of 4 on the enzyme; chloride binds to the enzyme when this group is protonated with a dissociation constant of 60 microM. The cyanide affinity of the enzyme is also regulated by the group with a pKa value of 4, but in this case cyanide binds to the unprotonated enzyme with a dissociation constant of 0.6 microM; only the protonated, uncharged form of cyanide reacts with the enzyme. Cyanide binding was competitively inhibited by chloride, and chloride binding was also competitively inhibited by cyanide. The EPR spectrum of the resting enzyme exhibited a rhombic high spin signal at g = 6.65, 5.28, and 1.97 with a low spin signal at g = 2.55, 2.32, and 1.82. Upon formation of the chloride complex, the spectrum was replaced with a new high spin EPR signal with g-values of 6.81, 5.04, and 1.95. The cyanide complex showed a low spin EPR signal with g-values of 2.83, 2.25, and 1.66. Examination of the enzymatic activity of the spleen green hemeprotein by following the chlorination of monochlorodimedon has indicated that the enzyme has the same chlorinating activity as myeloperoxidase; the spleen green peroxidase can catalyze the formation of hypochlorous acid from hydrogen peroxide and chloride ion. Comparison of the present data with those of myeloperoxidase has led to the conclusion that the structure of the iron center and its vicinity in spleen green hemeprotein is very similar, if not identical, to that of myeloperoxidase. The spleen enzyme can thus be used as a model to study the active center, and its environment, in myeloperoxidase.     

Activated conformers of Escherichia coli sulfite reductase heme protein subunit

The heme protein subunit of Escherichia coli sulfite reductase shows enhanced reactivity with its substrate and a number of other ligands after a cycle of reduction and reoxidation at alkaline pH. At pH 9.5 this variant of the enzyme possesses at least four EPR-detectable, chloride-sensitive high-spin conformers, in contrast to the single chloride-insensitive species observed in the oxidized, resting enzyme at pH 7.7. Quantitative reversal of the spectral and ligand-binding properties of the "activated" enzyme to those of the resting enzyme is observed on reacidification to pH 7.7. At intermediate pH values, there occurs an acid-catalyzed relaxation of the activated enzyme to the resting form. This reaction is distinct from the one responsible for the accelerated ligand binding and production of multiple EPR conformers, which appears to be regulated by a process with a pK of 8.5.     

Effect of disruption by sonication under different conditions on the activity of glucose dehydrogenase from resting spores of Bacillus subtilis

The activity of glucose dehydrogenase present in resting spores of Bacillus subtilis varied strikingly with the conditions for disrupting the spores by sonic treatment, namely, the time and strength of sonication, and the type and pH of the solution used for suspending the spores. When the resting spores were sonicated for 30 min at a current of 1.45 A in 100 mM phosphate buffer in the range of pH 6.0 to 6.6 or in deionized water, the enzyme activity of the former suspension was approximately 10 times higher than that of the latter suspension. However, the enzyme activity of the latter was markedly stimulated in the presence of sodium chloride. The glucose dehydrogenase from resting spores disrupted in 100 mM phosphate buffer (pH 6.6) was a salt-independent, active enzyme with a molecular weight of about 120,000, whereas the enzyme from resting spores disrupted in deionized water was a salt-dependent, inactive one with a molecular weight of about 55,000. A high concentration of dipicolinic acid strongly inhibited activation by a salt of inactive glucose dehydrogenase from resting spores in deionized water, suggesting one of its several important roles in vivo.     

A study of ligand binding to spleen myeloperoxidase

The ligand binding properties of spleen myeloperoxidase, a peroxidase formerly called "the spleen green hemeprotein", were studied as functions of temperature and pH, using chloride and cyanide as exogenous ligands. Ligand binding is influenced by a proton dissociable group with a pKa of 4. The protonated, uncharged form of cyanide binds to the unprotonated form of the enzyme, while chloride ion binds to the enzyme when this group is protonated. In both cyanide and chloride binding, the pH-dependent change in the apparent ligand affinity is due to a change in the apparent association rate with pH. The proton dissociable group on the enzyme involved in ligand binding has a delta H value of about 8 kcal . mol-1. The present results suggest that this ionizable group is the imidazole group of a histidine residue located near the ligand binding site.     

Paradoxical effects of methylmercury on the kinetics of cytochrome c oxidase

A stoichiometric amount of methylmercuric chloride substantially inhibits cytochrome c oxidase function under steady-state turnover conditions, where the enzyme is using its substrates, cytochrome c and oxygen, rapidly and continuously. Under these conditions, a reduction in activity of approximately 40% is observed. This is in accord with the results of Mann and Auer [Mann, A.J., & Auer, H.E. (1980) J. Biol. Chem. 255, 454-458], who used mercuric chloride and ethylmercuric chloride. Paradoxically, we found that addition of methylmercuric chloride can increase the activity of cytochrome c oxidase during its initial substrate utilization. This rate enhancement, measured under conditions where the enzyme cycles only a few times, is maximal for the resting state of the enzyme. "Pulsed" cytochrome c oxidase (i.e., enzyme that has been recently reduced and reoxidized) is considerably activated with respect to the resting enzyme, showing faster turnover rates (Antonini, 1977; Brunori et al., 1979). No significant rate enhancement upon treatment with methylmercuric chloride is seen in initial substrate utilization if the enzyme is pulsed immediately before the assay. The apparently contradictory effects of methylmercuric chloride on the resting and pulsed states of the oxidase under low turnover conditions may be reconciled by a model in which mercurial binding greatly stabilizes the enzyme in a state resembling that of the pulsed enzyme. A decrease in conformational flexibility may be the basis of the mercurial-induced diminution in activity of the enzyme during steady-state turnover conditions.     

Sulfate potentiation of the chloride activation of angiotensin converting enzyme

The angiotensin converting enzyme (ACE)-catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions, notably chloride. This activation is enhanced by sulfate; at pH 7.5, the effect is maximal at 0.8 M sulfate and is mediated through a specific interaction of the divalent anion with the enzyme, not through an increase in ionic strength. Sulfate decreases the apparent binding constant for chloride which manifests as a decrease of the apparent KM value, but it does not change kcat. Thus, at pH 7.5, sulfate solely affects substrate binding in accord with the ordered bireactant mechanism of chloride activation that pertains with this substrate [Bünning, P., & Riordan, J.F. (1983) Biochemistry 22, 100-116]. Increasing the pH from 6 to 9 in the absence of sulfate increases the apparent binding constant for chloride almost 60-fold from 3.3 to 190 mM. In the presence of 0.8 M sulfate, however, the change is only about 6-fold, from 0.7 to 4.2 mM. Over the same pH range, the apparent KM for furanacryloyl-Phe-Gly-Gly obtained with saturating chloride concentrations shifts from 0.14 to 0.48 mM, while in the presence of 0.8 M sulfate about 3-fold lower apparent KM values are obtained. Sulfate does not appear to affect the pK of a group on the enzyme that controls the mechanism of chloride activation but rather decreases the apparent KM by reducing the apparent binding constant for chloride.     

Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 1. The influence of substrate binding to reduced trimethylamine dehydrogenase on the intramolecular electron transfer between its prosthetic groups.

The trimethylamine dehydrogenase of bacterium W3A1 is reduced with the formation of a triplet state in which two electrons, derived from the substrate, are distributed between the [4Fe-4S] cluster and 6-S-cysteinyl-FMN semiquinone. In titration experiments at pH 8.5 about 1.0 mol of dimethylamine or 0.5 mol of trimethylamine per mol of the enzyme is required to titrate the enzyme to an endpoint. At pH values less than 8.0, however, an excess of trimethylamine is required to obtain maximal yield of the g = 4 e.p.r. signal, characteristic of the triplet state, or maximal absorbance at 365 nm which indicates formation of the flavin semiquinone. The binding of 0.86 mol of trimethylamine per mol of the enzyme could be detected by a gel chromatographic method. When the enzyme is titrated with dithionite in the presence of tetramethylammonium chloride, an endpoint is reached after the uptake of two electrons which give rise to the triplet state, whereas three electrons are consumed in the absence of tetramethylammonium chloride to reduce the enzyme completely. The enzyme is inhibited noncompetitively by tetramethylammonium chloride and the slopes of double reciprocal plots are a concave upwards function of inhibitor concentration. The data indicate the presence of a binding site for the substrate and other amines on the reduced enzyme which enhances the proportion of enzyme in the triplet state.     

Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme

The amidase activity of human alpha-thrombin has been studied in the pH range 5.5 to 10, and at four different chloride concentrations from 5 mM to 1 M. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over the pH range studied, with a minimum around pH 8. The pH dependence of the catalytic constant, kcat, shows multiple inflection points especially at low (less than 0.1 M) chloride concentrations, thereby implicating the existence of multiple catalytic forms of the enzyme. A general linkage scheme is proposed for the analysis of the effect of protons on thrombin amidase activity, and experimental data have globally been analysed over the entire pH range in terms of such a scheme. Four proton-linked ionizable groups seem to be involved in the control of thrombin amidase activity. Two of these groups change their pK value upon substrate binding to the enzyme and account for the pH dependence of Km. All four groups control the catalytic activity of the enzyme which decreases with increasing protonation. Chloride has little effect on Km, while kcat changes significantly at pH less than 8. This effect is due to an increased enzymatic activity of the highly protonated intermediates at high chloride concentrations, as well as to the pK shift of two proton-linked ionizable groups.     

Cyanide stimulated dissociation of chloride from the catalytic center of oxidized cytochrome c oxidase

A comparison of bovine cytochrome c oxidase isolated in the presence and the absence of chloride salts reveals that only enzyme isolated in the presence of chloride salts is a mixture of a complex of oxidized enzyme with chloride (CcO.Cl) and chloride-free enzyme (CcO). Using a spectrophotometric method for chloride determination, it was shown that CcO.Cl contains one chloride ion that is released into the medium by a single turnover or by cyanide binding. Chloride is bound slowly within the heme a(3)-Cu(B) binuclear center of oxidized enzyme in a manner similar to the binding of azide. The pH dependence of the dissociation constant for the formation of the CcO.Cl complex reveals that chloride binding proceeds with the uptake of one proton. With both forms of the enzyme the dependence of the rate of reaction for cyanide binding upon cyanide concentration asymptotes a limiting value indicating the existence of an intermediate. With CcO.Cl this limiting rate is 10(3) higher than the rate of the spontaneous dissociation of chloride from the binuclear center and we propose that the initial step is the coordination of cyanide to Cu(B) and in this intermediate state the rate of dissociation of chloride is substantially enhanced.     

Activation of rabbit muscle fructose 1,6-bisphosphatase by histidine and carnosine

Histidine and its derivatives increased rabbit muscle fructose 1,6-bisphosphatase activity at neutral pH with positive cooperativity. In the presence of histidine and carnosine the optimum pH shifted from pH 8.0 to 7.4. The cooperative response of the enzyme to AMP and fructose 1,6-bisphosphate was observed in the presence of the histidine derivatives. Of a number of divalent cations tested, only Zn2+ was found to be an effective inhibitor of enzyme activity at low concentrations. The kinetic data suggested that Zn2+ acted as inhibitor as well as activator for the enzyme activity; a high affinity binding site was associated with Ki of approximately 0.5 microM Zn2+ and a catalytic site was associated with Km of approximately 10 microM Zn2+. Rabbit muscle fructose 1,6-bisphosphatase bound 4 equivalents of Zn2+/mol, presumably 1 per subunit, in the absence of fructose 1,6-bisphosphate. Two equivalents of Zn2+/mol bound to the enzyme were readily removed by dialysis or gel filtration in the absence of a chelating agent. The other two equivalents of Zn2+/mol were removed by histidine and histidine derivatives of naturally occurring chelators with concomitant increase in activity.     

Isoionic titration and isopycnic density gradient centrifugation studies of magnesium activation and subunit dissociation in yeast enolase.

Isopycnic density gradient centrifugations were performed on yeast enolase A in cesium chloride and sulfate at the isoionic pH (6.0–6.5) and at pH 8.1. The dissociation of the enzyme appears to be greater at the more alkaline pH. No large effect of the cofactor magnesium or pH or dissociation on the isopycnic point was found. Isoionic titrations were carried out in the presence and absence of magnesium using both salts. The metal reduces net anion binding by isoionic enzyme and net cation binding by pH 8.1 enzyme by about 1.6 – 3.4 equivalents at 0.05 ionic strength and somewhat less at the isopycnic ionic strengths. It is concluded that the metal does not significantly affect net hydration of the enzyme and that subunit dissociation is not accompanied by large changes in hydration or salt binding.     

Probing the role of the chloride ion in the mechanism of human pancreatic alpha-amylase.

Human pancreatic alpha-amylase (HPA) is a member of the alpha-amylase family involved in the degradation of starch. Some members of this family, including HPA, require chloride for maximal activity. To determine the mechanism of chloride activation, a series of mutants (R195A, R195Q, N298S, R337A, and R337Q) were made in which residues in the chloride ion binding site were replaced. Mutations in this binding site were found to severely affect the ability of HPA to bind chloride ions with no binding detected for the R195 and R337 mutant enzymes. X-ray crystallographic analysis revealed that these mutations did not result in significant structural changes. However, the introduction of these mutations did alter the kinetic properties of the enzyme. Mutations to residue R195 resulted in a 20-450-fold decrease in the activity of the enzyme toward starch and shifted the pH optimum to a more basic pH. Interestingly, replacement of R337 with a nonbasic amino acid resulted in an alpha-amylase that no longer required chloride for catalysis and has a pH profile similar to that of wild-type HPA. In contrast, a mutation at residue N298 resulted in an enzyme that had much lower binding affinity for chloride but still required chloride for maximal activity. We propose that the chloride is required to increase the pK(a) of the acid/base catalyst, E233, which would otherwise be lower due to the presence of R337, a positively charged residue.     

Haloalkane dehalogenases: steady-state kinetics and halide inhibition

The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.     

Biologically active polymer supports based on cellulosic derivatives: synthesis and kinetic study

Bioactive cellulose derivatives have been synthesised by coupling enzymes/antibiotics on carboxymethyl cellulose acid chloride and cellulose carbonate. The effect of pH and temperature on the enzymatic activity of amyloglucosidase immobilised on cellulose carbonate was studied. Michaelis-Menten kinetics have been obeyed to the first degree of approximation despite the restricted mobility of the attached enzyme on the polymer support. Lineweaver-Burk plots for the amyloglucosidase immobilized on carboxymethyl cellulose acid chloride at ambient pH with cellulose carbonate at pH 8 have also been plotted. The Michaelis-Menten constant for the immobilized amyloglucosidase on cellulose carbonate at pH 8 was 9.1 mM, and the activation energy for starch hydrolysis was 21.8 kcals/mole.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

Inhibition and recognition studies on the glutathione-binding site of equine liver glutathione S-transferase.

Equine liver glutathione S-transferase has been shown to consist of two identical subunits of apparent Mr 25,500 and a pl of 8.9. Kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene as a substrate suggests a random rapid-equilibrium mechanism, which is supported by inhibition studies using glutathione analogues. S-(p-Bromobenzyl)glutathione and the corresponding N alpha-, CGlu- and CGly-substituted derivatives have been found, at pH 6.5, to be linear competitive inhibitors, with respect to GSH, of glutathione transferase. N-Acetylation of S-(p-bromobenzyl)glutathione decreases binding by 100-fold, whereas N-benzoylation and N-benzyloxycarbonylation abolish binding of the derivative to the enzyme. The latter effect has been attributed to a steric constraint in this region of the enzyme. Amidation of the glycine carboxy group of S-(p-bromobenzyl)glutathione decreases binding by 13-fold, whereas methylation decreases binding by 70-fold, indicating a steric constraint and a possible electrostatic interaction in this region of the enzyme. Amidation of both carboxy groups decreases binding significantly by 802-fold, which agrees with electrostatic interaction of the glutamic acid carboxy group with a group located on the enzyme.     

Release of proteinase from mycelium of Mucor hiemalis

When Mucor hiemalis NRRL 3103 was grown in soybean medium, only a small fraction of the proteinase produced by the organism appeared in the culture filtrate, whereas the bulk of the enzyme was bound to the mycelial surface. Optimal pH of the proteinase ranged from 3.0 to 3.5. Inclusion of sodium chloride or other ionizable salts in the growth medium, however, resulted in the liberation from the mycelium of the loosely bound enzyme as it was formed. Maximal release of proteinase was achieved at a sodium chloride concentration of 0.5 m. The loosely bound proteinase was eluted also from intact resting mycelium by ionizable salts but not by water or by nonionizable substances. The amount of enzyme eluted from the mycelium depended upon the concentration of sodium chloride up to 0.3 m. Since liberation took place rapidly even at 0 C, a loose ionic linkage must exist rather than a biochemical binding of the enzyme to the mycelium. The recovery of proteolytic activity from repeated salt extractions was greater than that originally detected in the intact mycelium, possibly owing to unmasking of more active enzymes or functional groups. Further proteinase activity was released when salt-extracted mycelium was ruptured. Part of the proteinase thus observed was firmly attached to the cell fraction, and part of it appeared in the supernatant fluid. These conditions implied the presence of intracellular or firmly attached proteinase which could be partially released.     

Manganese-containing superoxide dismutase from Escherichia coli: reversible resolution and metal replacements.

Exposure of the manganese-containing Superoxide dismutase of Escherichia coli to pH 3.2, in the presence of 0.7 m guanidinium chloride, causes a rapid loss of manganese and of activity. The apoenzyme so produced can be reconstituted by addition of MnCl₂ followed by neutralization. In contrast, manganese cannot be restored to the apoenzyme by adding MnCl₂after neutralization. The reconstituted enzyme is indistinguishable from the native enzyme in terms of its catalytic activity or electrophoretic behavior on polyacrylamide gels. Co(II), Ni(II), Zn(II), Fe(II), or Cu(II) could compete with Mn(II) during reconstitution of the apoenzyme. In the cases of Co(II), Ni(II), and Zn(II), it was shown that, in preventing reconstitution by Mn(II), they were themselves bound to the enzyme in stoichiometric amounts, in place of Mn(II). The binding of Fe(II) was also explored and was distinct in that the enzyme could bind more than stoichiometric amounts of this metal. None of the derivatives, in which Mn(II) had been replaced by another metal, were catalytically active. Nevertheless, these derivatives could be again resolved by exposure to acid guanidinium chloride and could then be converted back into the active holoenzyme by neutralization after addition of MnCl₂. It appears that the active site of this enzyme can accommodate and can tightly bind several metals other than manganese, but exhibits activity only with manganese. It also appears that movement of metal out of or into this site is only feasible at low pH and in the presence of a chaotropic agent. A substantial amount of the cobalt-substituted enzyme was prepared and its optical properties were recorded.     

Pepsin immobilized by covalent fixation to hydroxyalkyl methacrylate gels: preparation and characterization.

Insoluble active derivatives of pepsin (EC 3.4.23.1) were prepared by covalent binding of this enzyme to hydroxyalkyl methacrylate gels modified with 1,6-diaminohexane or epsilon-aminocaproic acid in an acid medium by means of water-soluble carbodiimide. The amount of attached enzyme, its proteolytic activity, pH activity curves of the preparations obtained and the time and pH dependence of their stability were determined.