Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase

Calcineurin, a calmodulin-regulated phosphatase, is composed of two distinct subunits (A and B) and requires certain metal ions for activity. The binding of the two most potent activators, Ni2+ and Mn2+, to calcineurin and its subunits has been studied. Incubation of the protein with 63Ni2+ (or 54Mn2+) followed by gel filtration to separate free and protein-bound ions indicated that calcineurin could maximally bind 2 mol/mol of Ni2+ or Mn2+. While isolated A subunit also bound 2 mol/mol of Ni2+, no Mn2+ binding was demonstrated for either isolated A or B subunit. When bindings were monitored by nitrocellulose filter assay, only 1 mol/mol bound Ni2+ or Mn2+ was detected, suggesting that the two Ni2+ (or Mn2+) binding sites had different relative affinities and that only metal ions bound at the higher affinity sites were detected by the filter assay. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the filter assay-measured Ni2+ (or Mn2+) binding by only 30%. Preincubation of the protein with Zn2+ decreased the filter assay-measured Ni2+ or Mn2+ binding by 90 or 17%, respectively. The results suggest that the higher affinity sites are a Ni2+-specific site and a distinct Mn2+-specific site. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the gel filtration-determined Ni2+ (or Mn2+) binding from 2 to 1 mol/mol suggesting that calcineurin also contains a site which binds either metal ion. The time course of Ni2+ (or Mn2+) binding was correlated with that of the enzyme activation, and the extent of deactivation of the Ni2+-activated calcineurin by EDTA or by incubation with Ca2+ and calmodulin (Pallen, C. J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141) was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation.     

Zinc inhibits calcineurin activity in vitro by competing with nickel

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.     

Biochemical and kinetic characterization of the DNA helicase and exonuclease activities of werner syndrome protein

The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg(2+) and ATP with a K(m) of 140 microm for the ATP-Mg(2+) complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg(2+) concentration. Mn(2+) and Ni(2+) substituted for Mg(2+) as a cofactor for WRN helicase, whereas Fe(2+) or Cu(2+) (10 microm) profoundly inhibited WRN unwinding in the presence of Mg(2+).Zn(2+) (100 microm) was preferred over Mg(2+) as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn(2+) strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn(2+) binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding.     

Metal cofactor requirement of β-lactamase II

1. The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn(2+)-containing enzyme. 2. Removal of Zn(2+) led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn(2+) by Co(2+), Cd(2+), Mn(2+) or Hg(2+) gave enzymes with significant, but lower, beta-lactamase activity. No activity was detected in the presence of Cu(2+), Ni(2+), Mg(2+) or Ca(2+). 4. Equilibrium dialysis indicated that the enzyme had at least two Zn(2+) binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn(2+) indicated that activity paralleled binding of Zn(2+) to the site of highest affinity. 5. Replacement of Zn(2+) by Co(2+) and Cd(2+) gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured beta-lactamase II with Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce beta-lactamase II activity in high yield.     

Doxorubicin inhibits the phosphate-transport protein reconstituted in liposomes. A study on the mechanism of the inhibition

Using Thr(P)-inhibitor-1 and Ser(P)-casein as substrates, studies on the activation of calcineurin purified from bovine brain have been carried out. The phosphatase requires the synergistic action of Ca2+, calmodulin and another divalent cation (Mg2+, Mn2+, Co2+ or Ni2+, but not Zn2+) for full expression of its activity. Ca2+ and Ca2+ X calmodulin act as allosteric activators to transform the phosphatase to a relaxed conformation, while Mg2+ acts solely as a cofactor for the catalytic action of the enzyme. In addition to their function as cofactors for catalysis, transition metal ions can also substitute for Ca2+ as allosteric activators. Ca2+ and calmodulin exert their activating effects mainly by increasing the Vm of the phosphatase reaction with little effect on the Km values for the substrates or on the KA values for the divalent cation cofactors. The predominant factor in dictating the catalytic properties of calcineurin is the divalent cation cofactor. For example, with Mg2+ as a cofactor, the phosphatase exhibits an optimum around pH 8.0-8.5; while with a transition metal ion as a cofactor, the optimum is around pH 7.0-7.5, regardless of whether Thr(P)-inhibitor-1 or Ser(P)-casein serves as a substrate, in the absence or the presence of Ca2+ X calmodulin.     

Zinc inhibition of cAMP signaling.

Zn(2+) is required as either a catalytic or structural component for a large number of enzymes and thus contributes to a variety of important biological processes. We report here that low micromolar concentrations of Zn(2+) inhibited hormone- or forskolin-stimulated cAMP production in N18TG2 neuroblastoma cells. Similarly, low concentrations inhibited hormone- and forskolin-stimulated adenylyl cyclase (AC) activity in membrane preparations and did so primarily by altering the V(max) of the enzyme. Zn(2+) also inhibited recombinant isoforms, indicating that this reflects a direct interaction with the enzyme. The IC(50) for Zn(2+) inhibition was approximately 1-2 microm with a Hill coefficient of 1.33. The dose-response curve for Zn(2+) inhibition was identical for AC1, AC5, and AC6 as well as for the C441R mutant of AC5 whose defect appears to be in one of the catalytic metal binding sites. However, AC2 displayed a distinct dose-response curve. These data in combination with the findings that Zn(2+) inhibition was not competitive with Mg(2+) or Mg(2+)/ATP suggest that the inhibitory Zn(2+) binding site is distinct from the metal binding sites involved in catalysis. The prestimulated enzyme was found to be less susceptible to Zn(2+) inhibition, suggesting that the ability of Zn(2+) to inhibit AC could be significantly influenced by the coincidence timing of the input signals to the enzyme.     

Glycerolipid biosynthesis in rat adipose tissue. I. Properties and distribution of glycerophosphate acyltransferase and effect of divalent cations on neutral lipid formation

A sensitive radioactive assay of acyl CoA:sn-glycerol-3-phosphate-O-acyltransferase (EC 2.3.1.15) was developed to study the properties and subcellular distribution of this enzyme in rat epididymal adipose tissue. The esterification of sn-glycerol-3-phosphate was measured in the presence of palmitoyl CoA or palmitate, ATP, CoA, and Mg(2+) at pH 7.5. The presence of glycerophosphate acyltransferase was detected in both mitochondria and microsomes. The product of this reaction was identified as phosphatidate by thin-layer chromatography and dual isotope incorporation studies. Several divalent cations reduced the activity of this enzyme. Although Mg(2+) was not required for the activity of glycerophosphate acyltransferase, its addition to the incubation mixture resulted in an increased formation of neutral lipids at the expense of phosphatidate. This result is explained by an activation of microsomal phosphatidate phosphatase (EC 3.1.3.4). The effect of Mg(2+) was completely abolished by Ni(2+), Co(2+), Mn(2+), and Zn(2+). These studies suggest that the balance between Mg(2+) and several other divalent ions may be important in the regulation of neutral lipid synthesis in adipose tissue.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Calcineurin immunoprecipitated from bovine brain extract contains no detectable Ni2+ or Mn2+

Purified calcineurin phosphatase is converted upon incubation in millimolar Ni2+ or Mn2+ to an active form by association with these metal activators. The bound metal ion is not dissociable from calcineurin by dialysis or gel filtration, but can be released upon prolonged incubation of the enzyme with Ca2+/calmodulin or chelating agents (Pallen, C.J., and Wang, J.H. (1986) J. Biol. Chem. 261, 16115-16120). The present study has been undertaken to test the possibility that calcineurin in brain may contain tightly bound Ni2+ or Mn2+. A monoclonal antibody (VA1) immunoaffinity matrix was prepared and shown to affect specific precipitation of calcineurin from crude bovine brain extract. Using [3H]-, [63Ni2+]-, and [54Mn2+]calcineurin added to the extract as radioactive tracer, it was found that up to 80% of the calcineurin could be immunoprecipitated, and that more than 50% of the originally bound metal ions could be detected in the immunoprecipitate. When samples of calcineurin immunoprecipitated from brain extracts were analyzed by atomic absorption spectroscopy, Ni2+ and Mn2+ were not detected, whereas, Zn2+, a constitutive metal of calcineurin (King, M. M., and Huang, C. Y. (1984) J. Biol. Chem. 259, 8847-8856) was found in the expected amount. The result suggests that calcineurin in brain does not contain tightly associated Ni2+ or Mn2+.     

Allosteric modulation of Euphorbia peroxidase by nickel ions

A class III peroxidase, isolated and characterized from the latex of the perennial Mediterranean shrub Euphorbia characias, contains one ferric iron-protoporphyrin IX pentacoordinated with a histidine 'proximal' ligand as heme prosthetic group. In addition, the purified peroxidase contained 1 mole of endogenous Ca(2+) per mole of enzyme, and in the presence of excess Ca(2+), the catalytic efficiency was enhanced by three orders of magnitude. The incubation of the native enzyme with Ni(2+) causes reversible inhibition, whereas, in the presence of excess Ca(2+), Ni(2+) leads to an increase of the catalytic activity of Euphorbia peroxidase. UV/visible absorption spectra show that the heme iron remains in a quantum mechanically mixed-spin state as in the native enzyme after addition of Ni(2+), and only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ni(2+). In the presence of H(2)O(2) and in the absence of a reducing agent, Ni(2+) decreases the catalase-like activity of Euphorbia peroxidase and accelerates another pathway in which the inactive stable species accumulates with a shoulder at 619 nm. Analysis of the kinetic measurements suggests that Ni(2+) affects the H(2)O(2)-binding site and inhibits the formation of compound I. In the presence of excess Ca(2+), Ni(2+) accelerates the reduction of compound I to the native enzyme. The reported results are compatible with the hypothesis that ELP has two Ni(2+)-binding sites with opposite functional effects.     

Calmodulin antagonists differentiate between Ni(2+)- and Mn(2+)-stimulated phosphatase activity of calcineurin.

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazolium, a potent calmodulin antagonist, inhibited the Ni(2+)-stimulated calmodulin-independent phosphatase activity to much the same extent as it did the Ca2+/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni(2+)-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn(2+)-stimulated phosphatase activity and the other calmodulin antagonists examined increased the Mn(2+)-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni(2+)-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5 microM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni(2+)-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn(2+)-stimulated phosphatase activity also recovered from the inhibition by calmidazolium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni(2+)- and Mn(2+)-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni(2+)-stimulated phosphatase activity.     

Response of recombinant calcineurin to metal ions,reduction-oxidation agents,and enzymatic modification

Recombinant calcineurin heterodimer with the full length delta-isoform of the catalytic subunit (CaN(500)) was expressed in insect cells using the baculovirus system and compared to native bovine brain enzyme in its response to divalent metal ions, redox reagents, and enzymatic modification of arginine residues. The response to various metal ions showed essentially the same profile as bovine brain calcineurin, although Co2+ and Zn2+ did not support recombinant activity as well. Kinetic analysis showed that metal ion and substrate binding were not independent, as found for the bovine brain calcineurin. Incubation with DTT or ascorbate alone caused similar effects on the activity of both enzymes, but different responses were observed when incubated with both DTT and ascorbate; only the recombinant enzyme showed activation. Arginine deimination of recombinant calcineurin by peptidylarginine deiminase resulted in the loss of 60-80% of its phosphatase activity with protection observed if calmodulin was present. Recombinant calcineurin was reactivated by treatment with the protease clostripain, suggesting that deimination of an arginine in the carboxyl terminal domain may be responsible for the loss of phosphatase activity and decreased calmodulin binding [Arch. Biochem. Biophys. 318 (1995) 370]. Supporting this conclusion, a truncated variant of the catalytic subunit lacking the carboxyl terminus showed no loss of phosphatase activity compared to full length calcineurin subunit and contained lower amounts of citrulline than the full length subunit after deimination. These different responses of recombinant calcineurin are consistent with conformational differences compared to bovine brain calcineurin and raise questions about its utility for studying the mechanism of calcineurin.     

Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii

The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.     

Purification and properties of α-d-mannosidase from rat epididymis

1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.     

Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein

Urea is the nitrogen fertilizer most utilized in crop production worldwide. Understanding all factors involved in urea metabolism in plants is an essential step towards assessing and possibly improving the use of urea by plants. Urease, the enzyme responsible for urea hydrolysis, and its accessory proteins, necessary for nickel incorporation into the enzyme active site and concomitant activation, have been extensively characterized in bacteria. In contrast, little is known about their plant counterparts. This work reports a detailed characterization of Glycine max UreG (GmUreG), a urease accessory protein. Two forms of native GmUreG, purified from seeds, were separated by metal affinity chromatography, and their properties (GTPase activity in absence and presence of Ni(2+) or Zn(2+), secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni(2+) and Zn(2+) was determined by isothermal titration calorimetry. rGmUreG binds Zn(2+) or Ni(2+) differently, presenting a very tight binding site for Zn(2+) (K (d) = 0.02 ± 0.01 μM) but not for Ni(2+), thus suggesting that Zn(2+) may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn(2+) stabilizes a dimeric form of the rGmUreG, while NMR measurements indicate that rGmUreG belongs to the class of intrinsically disordered proteins. A homology model for the fully folded GmUreG was built and compared to bacterial UreG models, and the possible sites of interaction with other accessory proteins were investigated.     

Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.     

Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli

The soluble pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors. The sth gene in Escherichia coli was cloned and expressed as a fused protein (EcSTH). The purified EcSTH displayed maximal activity at 35 °C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5 h at 50 °C. The enzyme was stable at 4 °C for 25 days. The apparent K(m) values of EcSTH were 68.29 μM for NADPH and 133.2 μM for thio-NAD(+) . The k(cat) /K(m) ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD(+) . Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD(+) . EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions: Mn(2+) , Co(2+) , Zn(2+) , Ni(2+) and Cu(2+) . However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn(2+) , Ni(2+) and Cu(2+) . The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations.     

Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699

The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory. The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1). The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.     

Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals

Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes.     

The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme

Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a time-dependent deactivation of the phosphatase was observed. The process followed first order kinetics and was dependent on the presence of both Ca2+ and calmodulin. The deactivation rate constant at pH 7.6 and 30 degrees C was 0.06 min-1, which was increased by the substrate, p-nitrophenylphosphate (Km = 11 mM), to 0.47 min-1. PPi and nucleotides inhibited the enzyme competitively and accelerated the deactivation. The first order rate constant was increased to 2.3 min-1 by PPi (Ki = 55 microM) and to 8.0 min-1 by ADP (Ki = 0.94 mM). A theory dealing with the deactivation (applicable to chemical modification, etc.) of an enzyme in the absence and presence of various ligands is presented. The deactivated enzyme remained bound to calmodulin and was not reactivated by dissociation-reassociation of the calcineurin-calmodulin complex. Calcineurin was found to contain covalently bound phosphate; however, no difference in its content was detected upon deactivation, indicating that self-dephosphorylation was not involved. The deactivation could be reversed, as well as prevented, by divalent metal ions such as Ni2+ and Mn2+. Atomic absorption spectroscopy revealed nearly stoichiometric amounts of tightly bound Fe and Zn (but little other ions) on purified calcineurin, which remained bound during the calmodulin-dependent deactivation; removal of tightly bound metals is, therefore, not the cause of deactivation. Our results indicate that calcineurin is a metallophosphatase and not simply a Ca2+- and calmodulin-stimulated enzyme. In addition to the nondissociable Zn and Fe and the Ca2+ bound to the B subunit and calmodulin, the enzyme requires a divalent metal ion for structural stability and full activity.