Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.     

Mechanism of action of Mg2+ and Zn2+ on rat placental alkaline phosphatase. I. Studies on the soluble Zn2+ and Mg2+ alkaline phosphatases.

Rat placental alkaline phosphatase (EC 3.1.3.1), a dimer of 135,000 daltons, is strongly activated by Mg2+. However, Zn2+ has to be present on the apoenzyme to obtain this activation. Mg2+ alone is unable to reconstitute functional active sites. Excess Zn2+ which competes for the Mg2+ site leads to a phosphatase with little catalytic activity at alkaline pH but with normal active sites at acidic pH as shown by covalent incorporation of ortho-[32P]phosphate. Two enzyme species with identical functional active sites have been reconstituted that only differ by the presence of Zn2+ or Mg2+ at the effector site. A mechanism is presented by which alkaline phosphatase activity of rat placenta would be controlled by a molecular process involving the interaction of Mg2+ and Zn2+ with the dimeric enzyme molecule.     

Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation.

Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly whereas the other one was phosphorylated much more slowly at pH 4.2. Half of the sites were shown to be reactive at pH 8.0. Quenching experiments have shown that only one of the two sites is phosphorylated at any instant; this result was confirmed by the stopped flow observation of a burst of only 1 mol of nitrophenol per mol of dimeric phosphatase in the pre-steady state hydrolysis of p-nitrophenyl phosphate. The half-of-the-sites reactivity observed for the native and for the Mg2+-stimulated enzyme indicates that the same type of complex, the monophosphorylated complex, accumulates under steady state conditions with both types of enzymes. Mg2+ binding to the native enzyme at pH 8.0 increases considerably the dephosphorylation rate of this monophosphorylated intermediate. A possible mechanism of Mg2+ stimulation is discussed.     

Metal ion-induced conformational changes in Escherichia coli alkaline phosphatase.

Ultraviolet difference spectra are produced by the binding of divalent metal ions to metal-free alkaline phosphatase (EC 3.1.3.1). The interaction of the apoprotein with Zn2+, Mn2+, Co2+ and Cd2+, which induce the tight binding of one phosphate ion per dimer, give distinctly different ultraviolet spectra changes from Ni2+ and Hg2+ which do not induce phosphate binding. Spectrophotometric titrations at alkaline pH of various metallo-enzymes reveal a smaller number of ionizable tyrosines and a greater stability towards alkaline denaturation in the Zn2+- and Mn2+-enzymes than in the Ni2+-, Hg2+- and apoenzymes. The Zn2+- and Mn2+-enzymes have CD spectra in the region of the aromatic transitions that are different from the CD spectra of the Ni2+-, Hg2+- and apoenzymes. Modifications of arginines with 2,3-butanedione show that a smaller number of arginine residues are modified in the Zn2+-enzyme than in the Hg2+-enzyme. The presented data indicate that alkaline phosphatase from Escherichia coli must have a well-defined conformation in order to bind phosphate. Some metal ions (i.e. Zn2+, Co2+, Mn2+ and Cd2+), when interacting with the apoenzyme, alter the conformation of the protein molecule in such a way that it is able to interact with substrate molecules, while other metal ions (i.e. Ni2+ and Hg2+) are incapable of inducing the appropriate conformational change of the apoenzyme. These findings suggest an important structural function of the first two tightly bound metal ions in enzyme.     

Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies.

The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.     

L-histidinol dehydrogenase, a Zn2+-metalloenzyme

The enzymatic activity of L-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 mM MnCl2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn2+/mol dimer, KD = 37 microM. ZnCl2, which prevented the MnCl2 stimulation, also bound to the enzyme, 1.2 g-atom/mol dimer, KD = 51 microM, and prevented Mn2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 M urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl2 or ZnCl2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl2 or ZnCl2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn2+, but which may bind Mn2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn2+ fulfills an essential structural and/or catalytic role.     

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.     

The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues.

Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

Cow's milk alkaline phospharase. II. Subunit structure, metalloproteic nature and kinetic parameters (author's transl)]

Alkaline phosphatase (EC 3.1.3.1) from cow's milk as a dimer comprising two identical or very similar subunits of about 85 000 molecular weight. The enzyme contains 4.9 +/- 0.6 gatoms of zinc per mol of protein. The essential kinetic properties are the same as those of other alkaline phosphatases: variation of pH optimum value, the lack of specificity, increase of Km and V with pH value. The phosphotransferase activity is enlarged, at constant concentration of acceptor, with an increasing concentration of donor. The small size of molecules and the presence of hydroxyls and amino groups increase the percentage of transfer phosphate. The phosphotransferase reaction is better with the D-isomer of serine and the enzyme possesses a more important affinity for the D-phosphoserine.     

Mechanism of action of Zn2+ and Mg2+ on rat placenta alkaline phosphatase. II. Studies on membrane-bound phosphatase in tissue sections and in whole placenta.

Alkaline phosphatase (EC 3.1.3.1) bound to trophoblastic cells in rat placenta is activated by Mg2+ and inhibited by Zn2+ in the same way as is found with partially purified soluble alkaline phosphatase in the same tissue (PetitClerc, C., Delisle, M., Martel, M., Fecteau, C. & Brière, N. (1975) Can. J. Biochem. 53, 1089-1100). In studies done with tissue sections (6-10 micron), it is shown that alkaline phosphatase activity and labelling of active sites by orthophosphate are lost during incubation with ethanolamine at pH 9.0. Addition of Mg2+ causes total recovery of catalytic activity and active sites labelling. Zn2+ displaces and replaces at the Mg2+ binding sites. The affinity for both ions is similar, and dissociation of Zn2+ from the enzyme is a very slow process, even in the presence of Mg2+. The Zn2+-alkaline phosphatase and Mg2+-alkaline phosphatase, which only differ by the ion bound to an apparent modulator site, have the same catalytic activity at pH less than 7.0, but the Zn2+ species has little activity at alkaline pH. Phosphorylation of the enzyme by orthophosphate indicates that with both enzyme species phosphoryl intermediate does not accumulate at alkaline pH. These results suggest that with orthophosphate, the phosphorylation step is rate determining for both enzymes, and that Zn2+ affects this step to a much greater extent. It is proposed that Zn2+ and Mg2+ regulate alkaline phosphatase in rat placenta. The concentration of both ions in maternal serum and placenta suggest that such a mechanism could exist in vivo.     

Trypsin-modified alkaline phosphatase. Formation of apoenzyme monomer and hybrid dimer

The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects.     

Role of metal ions in Escherichia coli alkaline phosphatase. A study of the metal-water interaction by nuclear relaxation rate measurements on water protons.

The binding of metal to alkaline phosphatase from Escherichia coli and the binding of water and orthophosphate to the Me-2+-enzyme binary complex have been examined by water proton relaxation rate (PRR) measurements. Titration of the three paramagnetic metals, Mn2+, Cu2+, and Co2+, into apoalkaline phosphatase and the titrations of apoenzyme into metal have been carried out. Analysis of the spin-lattice relaxation rates for these titrations and of Scatchard binding curves derived from these results, as well as EPR data, show four tight manganese sites, between two and three tight copper sites, or four cobalt sites per enzyme dimer of molecular weight 80,000. The multiple sites for each metal are indistinguishable by these magnetic resonance techniques. Both the spin-lattice- and spin-spin-relaxation rates exhibit a negative temperature coefficient, showing that these processes are not exchange-limited. From a frequency dependence study of T-1 and from the T-1:T-2 ratio measured at 220 MHz, correlation times from the water-enzyme complexes have been estimated. For H20-Mn-2+-alkaline phosphatase, gamma c equals 1.55 times 10-9 s; for H20-Cu-2+ -alkaline phosphatase, gamma c equals 1.82 times 10-s; and for the cobalt complex, gamma c equals 1.0 times 10-12 s at 4 degrees. Assuming 1 water molecule bound per metal site, these correlation times correspond to the following water-metal distances: gamma (A) is 4.0 A for Mn-2+-H20, 3.4 A for Cu-2+-H20, and 2.8 A for Co-2+-H20. Thus, water is shown to bind directly to the metal atoms of alkaline phosphatase. The correlation between the length of the water-metal bond and the relative activity of the various metalloenzymes support the importance of this binding in the monophosphoesterase reaction catalyzed by alkaline phosphatase. Addition of excess orthophosphate to any of the water-metalloenzyme complexes does not displace an exchangeable water molecule from the metal site. The Mn-PO-4 distance which we have reported earlier (Zukin, R.S., Hollis, D.P., and Gray, G.A. (1973) Biochem. Biophys. Res. Commun. 53, 238) to be 7.3 A is consistent with this finding and suggests a model in which Pi binds to Mn-2+-alkaline phosphatase through a water bridge.     

31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH.

31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase. Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex. The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.     

Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P_i to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.     

Purification of terminal riboadenylate transferase from calf thymus gland.

A poly(A) polymerase has been purified from the soluble protein fraction of calf thymus gland. The activity is cytoplasmic and nonparticulate. Mn-2+ATP is the preferred substrate. On the basis of disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels, gel filtration, and sedimentation velocity in sucrose gradients, the enzyme has a molecular weight of 62,000 and appears to consist of one polypeptide chain. The enzyme preparation is shown to be nearly homogeneous by disc gel electrophoresis and isoelectric-focusing. The activity has a pI of about 7.4. The specific activity of the enzyme is about 1700 mumol per hour per mg of protein, giving a turnover number of about 1800 mol of substrate per mol of enzyme min- minus 1. The activity is highly specific for ATP and is inhibited by other ribonucleoside triphosphates. It is sensitive to high levels of RNA-polymerase inhibitors. Km for oligoadenylate is 50 muM in the presence of Mn-2+ and 200 muM in Mg-2+ and equivalent Vmax is achieved with either metal ion. The initiator function may be filled by a variety of oligoribonucleotides having a free 3'-OH.     

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii.

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.     

Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization.

Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KCl, respectively. Both isoenzymes have a molecular weight of approximately 180,000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.     

Alkaline phosphatase of Thiobacillus thioparus. Partial purification and properties of the enzyme.

Soluble alkaline phosphatase from Thiobacillus thioparus cells was purified about 230-fold. The enzyme had a mol. wt. of 50 000 daltons, optimum pH at 10.5, and was heat-resistant in the presence of diethanolamine. Polyacrylamide-gel electrophoresis demonstrated contamination of the preparation with inactive proteins and the presence of two active bands. The enzyme activity was distinctly stimulated by increasing concentrations of Tris or diethanolamine. In the presence of glycine, 1 mM-Zn2+ enhanced the enzyme activity; in Tris or diethanolamine buffers the activity was stimulated by 1 mM-Mg2+ whereas Zn2+ had a strong inhibitory effect. Glycine at concentrations exceeding 25 mM also inhibited the enzyme. Specificity of the enzyme is fairly broad.     

Zinc inhibition of renin and the protease from human immunodeficiency virus type 1

We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.