Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.     

Factors affecting the zinc content of E. coli alkaline phosphatase

Through experiments with radioactively labeled EDTA, it has been shown that alkaline phosphatasc from E. coli has a high affinity for binding EDTA, requiring extensive dialysis for removal. This paper reviews the results of zinc analyses of E. coli alkaline phosphatase prepared in the presence and absence of EDTA. The presence of EDTA in most preparations of alkaline phosphatase accounts for previous overestimates of the zine content of the enzyme.With radioactively labeled EDTA, direct evidence for the binding of EDTA to the metal-free alkaline phosphatase is presented. It has been shown that the apoprotein binds two EDTA molecules rather strongly. Addition of four metal ions are necessary for reactivation of this EDTA-contaminated apoenzyme. However, when the EDTA-contaminated apoenzyme is subject for extensive dialysis and EDTA is removed, the addition of two zinc ions restores the enzyme activity completely.     

Effect of magnesium on the properties of zinc alkaline phosphatase.

Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.     

A probe of the active site acidity of carboxypeptidase A

The substrate analogue 2-(1-carboxy-2-phenylethyl)-4-phenylazophenol is a potent competitive inhibitor of carboxypeptidase A. Upon ligation to the active site, the azophenol moiety undergoes a shift of pKa from a value of 8.76 to a value of 4.9; this provides an index of the Lewis acidity of the active site zinc ion. Examination of the pH dependence of Ki for the inhibitor shows maximum effectiveness in neutral solution (limiting Ki = 7.6 X 10(-7) M), with an increase in Ki in acid (pK1 = 6.16) and in alkaline solution (pK2 = 9.71, pK3 = 8.76). It is concluded that a proton-accepting enzymic functional group with the lower pKa (6.2) controls inhibitor binding, that ionization of this group is also manifested in the hydrolysis of peptide substrates (kcat/Km), and that the identity of this group is the water molecule that binds to the active site metal ion in the uncomplexed enzyme (H2OZn2+L3). Reverse protonation state inhibition is demonstrated, and conventional concepts regarding the mechanism of peptide hydrolysis by the enzyme are brought into question.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyridoxal 5'-phosphate and analogs as probes of coenzyme-protein interaction in Baccillus alvei tryptophanase.

Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.     

Inhibition of human alkaline phosphatases by vanadate.

Orthovanadate was shown to be a potent competitive inhibitor (Ki less than 1 microM) of purified alkaline phosphatase from human liver, intestine of kidney. Inhibition was reversed and full enzymic activity restored in the presence of 1mM-adrenaline. Phosphate and vanadate competed for the same binding site on the enzyme.     

Inhibition of beta-amylase activity by calcium, magnesium and zinc ions determined by spectrophotometry and isothermal titration calorimetry

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH = 4.8 (sodium acetate 16 mM) and T = 300K. The Ki and enthalpy of binding for calcium (13.4, 13.1 mM and -14.3 kJ/mol), magnesium (18.6, 17.8mM and -17.7 kJ/mol) and zinc (17.5, 17.7 mM and -20.0 kJ/mol) were found by spectrophotometric and ITC methods respectively.     

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.     

Polyoxyethylene 9-lauryl ether-solubilized alkaline phosphatase: synergistic stimulation by zinc and magnesium ions.

1. Polidocanol-solubilized apoalkaline phosphatase could be stimulated either by zinc ions (Kd = 8.5 nM) or by magnesium ions alone (Kd = 3.8 microM). 2. Zinc and magnesium ions had synergistic effects on Polidocanol-solubilized apoalkaline phosphatase, leading to a fully active enzyme (700-800 U/mg). 3. Zinc ions inhibited non-competitively the Polidocanol-solubilized apoenzyme (Ki = 7.1 microM) by displacing magnesium ions from their binding sites. 4. A model for the action of zinc and magnesium ions on the modulation of the enzyme activity is proposed.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

Metal specificity is correlated with two crucial active site residues in Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P(i)). Since release of P(i) is the slow step in the mechanism at alkaline pH, the enhanced binding of P(i) in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P(i) in the HW_Co structure explains the observed increase in activity. These alterations in P(i) affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme.     

Inhibition of β-Amylase Activity by Calcium,Magnesium and Zinc Ions Determined by Spectrophotometry and Isothermal Titration Calorimetry

The inhibition effect of metal ions on beta amylase activity was studied. The inhibitor-binding constant (Ki) was determined by spectrophotometric and isothermal titration calorimetric (ITC) methods. The binding of calcium, magnesium and zinc ion as inhibitors at the active site of barley beta amylase was studied at pH=4.8 (sodium acetate 16?mM) and T=300?K. The Ki and enthalpy of binding for calcium (13.4, 13.1?mM and -14.3?kJ/mol), magnesium (18.6, 17.8?mM and -17.7?kJ/mol) and zinc (17.5, 17.7?mM and -20.0?kJ/mol) were found by spectrophotometric and ITC methods respectively.     

Uridine diphosphate galactose 4-epimerase: nucleotide and 8-anilino-1-naphthalenesulfonate binding properties of the substrate binding site.

Escherichia coli UDP-galactose 4-epimerase in its native form (epimerase.NAD) binds 8-anilino-1-naphthalenesulfonate (ANS) at one tight binding site per dimer with a dissociation constant of 25.9 +/- 2.1 micrometer at pH 8.5 and 27 degrees C. This appears to be the substrate binding site, as indicated by the fact that ANS is a kinetically competitive reversible inhibitor with a Ki of 27.5 micrometer and by the fact that ANS competes with UMP for binding to the enzyme. Upon binding at this site the fluorescence quantum yield of ANS is enhanced 185-fold, and its emission spectrum is blue shifted from a lambdamax of 515 to 470.nm, which suggests that the binding site is shielded from water and probably hydrophobic. Competitive binding experiments with nucleosides and nucleotides indicate that nucleotide binding at this site involves coupled hydrophobic and electrostatic interactions. The reduced form of the enzyme (epimerase.NADH) has no detectable binding affinity for ANS. The marked difference in the affinities of the native and reduced enzymes for ANS is interpreted to be a manifestation of a conformational difference between these enzyme forms.     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

The importance of aspartate 327 for catalysis and zinc binding in Escherichia coli alkaline phosphatase.

In order to investigate the function of Asp-327, a bidentate ligand of one of the zinc atoms in Escherichia coli alkaline phosphatase, and the importance of this zinc atom in catalysis, site-specific mutagenesis was used to convert Asp-327 to either asparagine or alanine. The 10(7)-fold decrease in the kcat/Km ratio observed for the Asp-327----Ala enzyme compared to the wild-type enzyme indicates that the side chain of Asp-327 is important for zinc binding at the M1 site. However, only one of the two carboxyl oxygens of Asp-327 is essential for zinc binding, since the Asp-327----Asn enzyme shows approximately the same hydrolysis activity as the wild-type enzyme. The fact that the enzymatic activity of this mutant enzyme shows a dependence on zinc concentration suggests that the other carboxyl oxygen or the negative charge on the side chain of Asp-327 is important in binding of the zinc at the M1 site. However, the zinc hydroxyl must still be appropriately positioned to attack the phosphoserine in the Asp-327----Asn enzyme; therefore, the negative charge and at least one carboxyl oxygen of the side chain are not directly involved in positioning or deprotonating the zinc hydroxyl. 31P NMR studies indicate that the Asp-327----Asn enzyme exhibits transphosphorylation activity at both pH 8.0 and pH 10.0, but at a reduced level compared to the wild-type enzyme. The biphasic production of 2,4-dinitrophenylate in the pre-steady-state kinetics of the mutant enzymes at pH 5.5 suggests that the breaking of the phosphoenzyme covalent complex is rate-limiting for both mutant enzymes. These results suggest that the main function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that it may be important in helping to stabilize the alcohol leaving group.     

Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor

The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.     

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.