Shift In pH Optimum Research Articles

Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex

The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S 0.5 for 2-oxoglutarate, and in an increase of S 0.5 for NAD. Changes in ionic strength over the range of 0.05–0.2 M have little, if any, effect on S 0.5 for CoA. The Hill coeffient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4–7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca 2+, increases in ionic strength lower the inhibitory effects of ATP. The S 0.5 i for ATP in both presence and absence of Ca 2+ was not affected by changes in ionic strength in the range of 0.1–0.2 M. In contrast, the a 0.5 for ADP in the absence of Ca 2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity.

Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective Inhibition of Duodenal and Jejunal Villous Cell Alkaline Phosphatase by the Duodenal Ulcerogen Cysteamine

We have found that cysteamine-HCl, a potent duodenal ulcerogen, after a single subcutaneous injection (30 mg/100 g body weight), inhibited villous cell duodenal and jejunal alkaline phosphatase (APase) under in vivo and under in vitro conditions. The duodenal and jejunal crypt-cell APase was not susceptible to cysteamine inhibition. Ileal APase from both the villous and the crypt cells was unaffected by cysteamine. Tissue-nonspecific APase from the kidney and liver was not affected by cysteamine either. The differences in tissue and cellular accumulation of cysteamine, the submolecular differences in APase molecules, and its anatomical localization in mucosal cells along the small intestine could explain the different degrees of susceptibility to cysteamine inhibition. The extent of duodenal APase inhibition by cysteamine was highly pH-dependent and varied from 5% to 85% within a pH range of 7.5-10.5. A shift in pH optimum from 9.6 to 9.3 was found in the presence of cysteamine. The inhibition of duodenal villous cell APase was greatly dependent on cysteamine concentration (Ki = 2.65 mM). At a fixed concentration of cysteamine it was not influenced by substrate concentration. Cysteamine did not change the Km value for duodenal APase but did decrease its Vmax to 46% and 15% of the controls when added in the assay or injected subcutaneously, respectively, indicating that the inhibition was of the linear, 'noncompetitive' type. Somehow cysteamine increased the requirement in the activation energy for substrate hydrolysis as well. The data indicate that macromolecular transformations could take place in the mucosal cells of duodenum after cysteamine administration.

Immobilization of β glucosidase aggregates in calcium alginate

A commercial preparation of β glucosidase obtained from Aspergillus niger was immobilized in calcium alginate, after enzyme crosslinking with a bifunctional agent, which increases its molecular size with the formation of enzymatic aggregates. The immobilized enzyme exhibited changes of activity profiles related to temperature and pH, in comparison to the free enzyme. However, there was no shift in optimum pH and temperature upon immobilization. The catalyst showed an improved thermal stability when compared with the free enzyme, yielding a half-life of 23 days, at 50°C. The free enzyme exhibited a 9-day half life, determined at the same temperature.

PH-Dependent Stimulation of Peptidylglycine α-Amidating Monooxygenase Activity by a Granule-Associated Factor*

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a granule-associated enzyme that catalyzes the production of alpha-amidated peptides from their glycine-extended precursors, a posttranslational modification often required for full biological activity. PAM activity in crude homogenates of bovine neurointermediate pituitary has an acidic pH optimum for the peptide substrate alpha-N-Ac-Tyr-Val-Gly. During purification, the pH optimum shifts, so that purified bovine (b)PAM exhibits an alkaline pH optimum for this substrate with virtually no activity below pH 6.5. A factor that restores the ability of purified bPAM to produce alpha-amidated products at pH 6 was identified. In rat anterior pituitary this factor (denoted SPAM for stimulator of PAM activity) was a soluble protein with a mol wt of 44 K by gel filtration; its stimulatory activity could be reduced or eliminated by trypsin digestion or boiling. SPAM stimulated PAM activity at acidic pH by increasing the apparent Vmax and decreasing the apparent Michaelis-Menten constant (Km) for the peptide substrate. Like PAM, SPAM activity is localized to the secretory granule. Levels of SPAM activity in various rat tissues correlated closely with levels of PAM activity, with the greatest amount of SPAM activity in atrium, anterior pituitary, and neurointermediate pituitary. The distribution of PAM and SPAM between soluble and membrane fractions also correlated closely. In AtT-20 cell lines transfected with a complementary DNA (cDNA) encoding the full-length bPAM precursor, both SPAM and PAM activities were increased compared to wild type cells; both activities were decreased in a cell line expressing an antisense rat (r)PAM mRNA. In marked contrast, an AtT-20 cell line transfected with a cDNA encoding a truncated, soluble form of bPAM had elevated levels of PAM activity, but levels of SPAM activity were not increased compared to wild-type cells. These results suggest that SPAM activity is closely linked to the expression of full-length PAM. The interaction of PAM and SPAM may represent a site for regulation of the synthesis of bioactive peptides, particularly at low intragranular pH.

Substrate-dependent shift of optimum pH in porcine pancreatic .alpha.-amylase-catalyzed reactions

Porcine pancreatic alpha-amylase (EC 3.2.1.1, abbreviated as PPA) hydrolyzes alpha-D-(1,4) glucosidic bonds in starch and amylose at random, and the optimum pH for the substrates is 6.9. The optimum pH, however, shifted to 5.2 for the hydrolytic reaction of low molecular weight oligosaccharide substrates such as p-nitrophenyl alpha-D-maltoside, gamma-cyclodextrin, maltotetaitol, and maltopentaitol. The optimum pH for the oligosaccharides consisting of more than five glucose residues, such as maltopentaose and maltohexaitol, was 6.9. From the analysis of the hydrolysates, it was clear that the shift of the optimum pH occurred only when the fifth subsite of PPA in the productive binding modes was occupied by a glucosyl residue of the substrates. The value of Km was independent of pH between 4 and 10 but that of kcat was dependent on pH. The pH profiles of kcat for the above substrates did not fit a simple bell-shaped curve predicted by a two-catalytic-group mechanism. Instead, they were well analyzed theoretically by three pK values and two intrinsic kcat values. Enthalpy changes for the three pK's (4.90, 5.35, and 8.55 at 30 degrees C) were determined from the temperature dependence of pH profiles for maltopentaitol and maltohexaitol to be 0.0, 2.87, and 7.33 kcal/mol, respectively. These results indicate that productive binding modes of the substrates directly affect the catalytic function of the enzyme. From the present thermodynamic analysis and reported three dimensional structure at the active site of PPA [Buisson, G. (1987) EMBO J. 6, 3909-3916], one can assume that a histidyl residue (101, 201, or 299) acts as a proton donor and two carboxyl groups (Asp 197, Glu 233, or Asp 300) act as proton donors or acceptors, and the productive binding mode covering the fifth subsite changes configurations between the catalytic residues and the glucosidic bond hydrolyzed and modulates kinetic parameters depending on pH.

Immobilized enzymes in reverse micelles: Studies with gel‐entrapped trypsin and α‐chymotrypsin in AOT reverse micelles

Trypsin and alpha-chymotrypsin were immobilized by gelentrapment in polyacrylamide cross-linked with N,N(1)-methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30-40% activity after 1 week. The enzymes obey Michaelis-Menten kinetics in the investigated concentration range with K(m) values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nalpha-benzoyl-L-arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., Z--Ala--Phe--Leu--NH(2)) using water-insoluble substrate has been performed with gelentrapped alpha-chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.

Entrapping of acid phosphatase in poly(2‐hydroxyethyl methacrylate) matrices: Preparation and kinetic properties

AbstractThe present work was undertaken to determine the influence of immobilization of acid phosphatase by entrapment in poly(2‐hydroxyethyl methacrylate) (poly‐HEMA) gels on the rate and duration of p‐nitrophenyl phosphate hydrolysis. This enzyme was selected as model system for the sake of comparison with other immobilization techniques studied previously making use of natural polymers. Under certain conditions, catalyst inactivation induced by chemical agents employed during polymerization was very low. Activity recovery in the enzymatically filled hydrogels depends on acid phosphatase loading and attains a maximum at 2 mg per gram of support. The use of low molecular weight linear polymers as matrix fillers is advisable. The best results were achieved with poly(ethylene oxide) PEG 1500, but PEG 6000 and poly(N‐vinylpyrrolidone) PVP 10 000 are also effective. Bovine serum albumin (BSA) is useless. The highest value of activity recovery obtained was 24% that of free enzyme. Roughly 30% of enzyme is retained in active form in the hydrogel and works with an efficiency ranging from 97% to 90% depending on average particle size. The kinetic parameters Km and Ea suggest the presence of a combined diffusive and enzymatic reaction‐controlling mechanism. Only a minor shift of optimum pH was observed. Both operational and storage stability are largely improved upon entrapping in hydrogels.

A stepwise approach to the understanding of extracellular enzyme activity in soil I. Effect of electrostatic interactions on the conformation of a β- d-glucosidase adsorbed on different mineral surfaces

The enzymatic activity of sweet almond β- d-glucosidase adsorbed on various mineral surfaces was studied. Our aim was to elucidate the mechanism responsible for the observed changes in catalytic activity. The results of the investigation are discussed with reference to the hypotheses generally proposed to explain the well-documented shift in optimal pH of the activity of adsorbed enzymes. By separate determinations of enzymatic activity in a mineral suspension and of its supernatant solution, and comparison with a control without mineral added, we obtained accurate measurements of the catalytic activity of the adsorbed enzyme alone. Different pH profiles of activity were found when the enzyme was adsorbed onto montmorillonite, kaolinite and goethite. The activity profiles were also found to vary with ionic strength, the pH at which enzyme adsorbed onto the mineral surface, and in the case of goethite, on the nature of the anions in the buffer. Our observations cannot be adequately explained by assuming a more acidic microenvironment at the mineral surface. We postulate that on some mineral surfaces a conformational change is induced in the adsorbed protein, which reduces its catalytic activity. We contend that such conformational changes are due primarily to electrostatic forces.

Characterization and purification of the membrane-bound ATPase of the archaebacterium Methanosarcina barkeri.

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.

Effect of immobilization on the physical and kinetic properties of soluble and insoluble trypsin–albumin polymers

Bovine trypsin was crosslinked to human serum albumin (HSA) with glutaraldehyde to form soluble and insoluble copolymers. The physical and kinetic properties of trypsin and trypsin-HSA polymers were compared. Trypsin was heat labile, retaining only 24% of its enzymic activity after heating for 5 min at 60 degrees C. In contrast, under the same condition both the soluble and insoluble trypsin-HSA polymers showed enhanced resistance to heat in-activation, retaining 81 and 100% of their original activities, respectively. The trypsin-HSA polymers also showed shifts in pH optima, an increase in activation energy, and a broadening of their pH stability profiles.

Regulation of arylsulphatases: Inhibition of arylsulphatase from Haliotis rufusensis by phosphoric esters and shift of optimal pH depending on temperature

1. 1. Arylsulphatase activity of Haliotis rufusensis has been tested at different temperatures and pH. This enzyme is characterized by an optimum temperature at +50°C. 2. 2. The maximum pH activity is depending on temperature; by increasing temperature from +20 to +50°C, a shift of pH maximum has been observed towards the acidic side. 3. 3. The enzyme activity was tested with nitrocatecholsulphate, synthetic substrate, and with natural substrates, cerebroside sulphate and ascorbate 2-sulphate. These natural substrates are “competitive” with respect to the synthetic substrate. 4. 4. Nitrophenylphosphate is a powerful inhibitor of this enzyme; this effect is pH dependent. Several phosphoderivatives, including inositol-phosphatides and myo-inositolphosphate, are also important inhibitors. 5. 5. Arylsulphatase from Haliotis, like those from Helix and Patella, is a glycoprotein (muramyl-glycoprotein). Its electrophoretic behaviour is similar to that of arylsulphatase A from human liver, but AgNO 3 or NaCl treatment are ineffective in differentiating A and B forms.

Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley ((1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245–257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N′- dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N′- ethylmaleimide , p- chloromercuribenzoate , orthovanadate, or ouabain. Sulfolobus ATPases differ from F 1 and other transport ATPases so far described.

Characterization of membrane‐bound MgATPases, purified by aqueous two‐phase partitioning, from young sugar beet roots

Membrane‐bound MgATPase activity from roots of young sugar beet (Beta vulgaris L. cv. Monohill) was investigated in a membrane fraction purified by partition in an aqueous polymer two‐phase system. After two steps of “washing” with fresh bottom phase (rich in dextran), the polyethylene glycol rich top phase (U3) was practically free of mitochondrial membranes (cytochrome oxidase), and the remaining MgATPase activity showed high substrate specificity for ATP. An optimum for the MgATPase activity was found at pH 7. The activation by Na+ or K+ was strongest on the acid side without any observable shift in pH optimum. Oligomycin had no effect, but vanadate strongly inhibited the U3 MgATPase and the K+ activation was lost. The complex activation pattern achieved by varying the Na+/K+ ratio at constant total concentration was interpreted as a synergistic (Na++ K+)‐activation. The U3 fraction MgATP‐ase activity showed a 4‐fold increase in the presence of 0.01% Triton X‐100 implying that the MgATPase activity is located in vesicles of which 75% or more are sealed with the ATP binding site on the inside. Comparison with the properties of plasma membrane. ATPases from other plants indicated that the U3 fraction MgATPase was mainly of plasma membrane origin.

Shift of optimal pH in arylsulphatase from mollusca depending on temperature

1. 1. Arylsulphatase activity of Helix pomatia and Patella vulgata have been tested at different temperatures. 2. 2. The maximum pH activity changes depending on temperature; by increasing temperature a shift of pH maximum has been observed in both arylsulphatases. 3. 3. The influence of 4-nitrophenylphosphate (4-NPP), a synthetic inhibitor phosphoester, has been also studied at different temperatures. 4. 4. Differences are evident between inhibited and non-inhibited arylsulphatase from Helix pomatia and Patella vulgata. 5. 5. By comparing arylsulphatase from Helix and Patella with that of human liver, arylsulphatase from Helix resembles arylsulphatase type A, but it is not inhibited by AgNO 3; arylsulphatase from Patella migrates faster than Helix (as human arylsulphatase B does), but is not inhibited by NaCl, inhibitor of arylsulphatase B.

Certain mouse strains are deficient in a kidney brush-border metallo-endopeptidase activity

Preparations of microvilli from kidneys of BALB/c mice contain an alkaline metallo-endopeptidase, meprin (metallo-endopeptidase from renal tissue). Certain genealogically related inbred mice are markedly deficient in meprin activity. The meprin-deficient strains (CBA/J and C3H/HeJ) exhibit normal levels of other brush-border enzymes: alkaline phosphatase, aminopeptidase M and another proteinase, a phosphoramidon-sensitive neutral endopeptidase. Meprin deficiency cannot be attributed to a shift in pH optimum and is unlikely to be due to the presence of endogenous inhibitors.

Restoration of lead-inhibited 5-aminolevulinate dehydratase activity in whole blood by heat, zinc ion, and (or) dithiothreitol.

We examined effects of heat, zinc, ion, and dithiothreitol in restoring the activity of lead-inhibited-5-aminolevulinate dehydratase (EC 4.2.1.24). The ratio of non-activated to activated activity produced by dithiothreitol correlated well with blood lead concentration among 35 lead workers. The individual effects of heat, zinc, or dithiothreitol differ from each other in the shift of pH optimum as well as in the extent to which activity is restored. Dual or triple combinations of these agents show additive or complementary restoration of activity. The combination of heat and zinc or zinc and dithiothreitol expands the range of optimum concentration of zinc in restoring activity. Using these combinations of agents, we can expect more accurate evaluation of lead exposure than by measuring only activation of zinc. Although dithiothreitol most powerfully restores activity, it restores not only the activity inhibited by lead exposure but also the activity removed by oxidation of SH-groups in the enzyme molecule.

Restoration of lead-inhibited 5-aminolevulinate dehydratase activity in whole blood by heat, zinc ion, and (or) dithiothreitol.

Abstract We examined effects of heat, zinc, ion, and dithiothreitol in restoring the activity of lead-inhibited-5-aminolevulinate dehydratase (EC 4.2.1.24). The ratio of non-activated to activated activity produced by dithiothreitol correlated well with blood lead concentration among 35 lead workers. The individual effects of heat, zinc, or dithiothreitol differ from each other in the shift of pH optimum as well as in the extent to which activity is restored. Dual or triple combinations of these agents show additive or complementary restoration of activity. The combination of heat and zinc or zinc and dithiothreitol expands the range of optimum concentration of zinc in restoring activity. Using these combinations of agents, we can expect more accurate evaluation of lead exposure than by measuring only activation of zinc. Although dithiothreitol most powerfully restores activity, it restores not only the activity inhibited by lead exposure but also the activity removed by oxidation of SH-groups in the enzyme molecule.

Effects of amino acids on Thiobacillus acidophilus. II. Threonine deaminase.

Biosynthetic L-threonine deaminase was partially purified 73-fold with a 60% recovery from Thiobacillus acidophilus by ammonium sulfate fractionation and by Sepharose 6B-C1 chromatography. The optimal pH for enzyme activity was between 9.0 and 10.0 and no optimal pH shift was observed in the presence of L-isoleucine, an inhibitor. The enzyme was effectively inhibited by L-isoleucine and showed homotropic interaction only in the presence of L-isoleucine. Kinetic studies indicate that there are at least two threonine binding sites and at least two isoleucine binding sites. The Km for threonine is 2.5 x 10(-3) M. The inhibition due to isoleucine is reversed by low concentrations of L-valine. L-Valine at high concentration acts as a substrate analogue and competitively inhibits L-threonine binding at the active site; the K1 is 1.6 x 10(-2) M.

The stimulation of Escherichia coli aspartate transcarbamylase activity by adenosine triphosphate : Relation with the other regulatory conformational changes; a model

The process of stimulation of Escherichia coli aspartate transcarbamylase activity by ATP was investigated. The efficiency of the phenomenon increases with the number of phosphate groups bound to adenosine. The pH dependence of the stimulation by ATP and adenylyl methylenediphosphonate indicates that the binding of these nucleotides requires the ionization of their last phosphate acidic group. The aspartate trans-carbamylase activity does not appear to be under the influence of the “energy charge ratio” but rather to depend directly on the ATP concentration. The stimulation decreases when the aspartate concentration increases. Saturating amounts of ATP do not provoke the shift in optimum pH for the catalytic activity which is shown to be associated with the homotropic co-operative interactions between the catalytic sites. This result provides additional evidence that homotropic and heterotropic interactions correspond to different molecular mechanisms. ATP reverses the effect of the feedback inhibitor CTP. A model is proposed to account for the relationship between the process of stimulation by ATP and the other regulatory conformational changes.