Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)6(3+) substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2'-fluoro modification at nt -1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E*S formation apart from the catalytic performance.     

Structural basis of the zinc inhibition of human tissue kallikrein 5

Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has trypsin-like substrate specificity, is inhibited by metal ions, and is abundantly expressed in human skin, where it is believed to play a central role in desquamation. To further understand the interaction of hK5 with substrates and metal ions, active recombinant hK5 was crystallized in complex with the tripeptidyl aldehyde inhibitor leupeptin, and structures at 2.3 A resolution were obtained with and without Zn2+. While the overall structure and the specificity of S1 pocket for basic side-chains were similar to that of hK4, a closely related family member, both differed in their interaction with Zn2+. Unlike hK4, the 75-loop of hK5 is not structured to bind a Zn2+. Instead, Zn2+ binds adjacent to the active site, becoming coordinated by the imidazole rings of His99 and His96 not present in hK4. This zinc binding is accompanied by a large shift in the backbone conformation of the 99-loop and by large movements of both His side-chains. Modeling studies show that in the absence of bound leupeptin, Zn2+ is likely further coordinated by the imidazolyl side-chain of the catalytic His57 which can, similar to equivalent His57 imidazole groups in the related rat kallikrein proteinase tonin and in an engineered metal-binding rat trypsin, rotate out of its triad position to provide the third co-ordination site of the bound Zn2+, rendering Zn2+-bound hK5 inactive. In solution, this mode of binding likely occurs in the presence of free and substrate saturated hK5, as kinetic analyses of Zn2+ inhibition indicate a non-competitive mechanism. Supporting the His57 re-orientation, Zn2+ does not fully inhibit hK5 hydrolysis of tripeptidyl substrates containing a P2-His residue. The P2 and His57 imidazole groups would lie next to each other in the enzyme-substrate complex, indicating that incomplete inhibition is due to competition between both imidazole groups for Zn2+. The His96-99-57 triad is thus suggested to be responsible for the Zn2+-mediated inhibition of hK5 catalysis.     

Human recombinant thiamine triphosphatase: purification, secondary structure and catalytic properties

Thiamine triphosphate (ThTP) is found in most living organisms and it may act as a phosphate donor for protein phosphorylation. We have recently cloned the cDNA coding for a highly specific mammalian 25 kDa thiamine triphosphatase (ThTPase; EC 3.6.1.28). As the enzyme has a high catalytic efficiency and no sequence homology with known phosphohydrolases, it was worth investigating its structure and catalytic properties. For this purpose, we expressed the untagged recombinant human ThTPase (hThTPase) in E. coli, produced the protein on a large scale and purified it to homogeneity. Its kinetic properties were similar to those of the genuine human enzyme, indicating that the recombinant hThTPase is completely functional. Mg2+ ions were required for activity and Ca2+ inhibited the enzyme by competition with Mg2+. With ATP as substrate, the catalytic efficiency was 10(-4)-fold lower than with ThTP, confirming the nearly absolute specificity of the 25 kDa ThTPase for ThTP. The activity was maximum at pH 8.5 and very low at pH 6.0. Zn2+ ions were inhibitory at micromolar concentrations at pH 8.0 but activated at pH 6.0. Kinetic analysis suggests an activator site for Mg2+ and a separate regulatory site for Zn2+. The effects of group-specific reagents such as Woodward's reagent K and diethylpyrocarbonate suggest that at least one carboxyl group in the active site is essential for catalysis, while a positively charged amino group may be involved in substrate binding. The secondary structure of the enzyme, as determined by Fourier-transform infrared spectroscopy, was predominantly beta-sheet and alpha-helix.     

Arylamidase activity of neutral proteinase from Saccharomonospora canescens. Comparison with other Zn-proteinases that exhibit the same activity

The arylamidase activity of Zn-proteinase from Saccharomonospora canescens (NPS) was studied with series of peptide nitroanilides of varying amino acid sequence and N-acyl blocking groups. The partial mapping of the enzyme S(1), S(2), S(3), S(4) subsites shows that variations in all positions P(1) to P(4) in the substrate structure affect the catalytic efficiency. The importance of P(4)-S(4) and P(1)-S(1) interactions, which is a characteristic feature of the serine proteinases, is evidenced for the studied Zn-proteinases NPS and serralysin too. The presence of arylamidase activity in the case of Zn-proteinases-astacin EC 3.4.24.21 and serralysin EC 3.4.24.40 is correlated with some specific characteristics of their active site structure: penta-coordinated Zn(2+) and a tyrosyl residue as a fifth ligand to the Zn(2+). It is assumed that this tyrosyl residue plays a role in the productive binding and stabilization of the tetrahedral adduct formed during the reaction of enzyme-catalysed hydrolysis of peptide arylamides of corresponding length and sequence.     

Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides.

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.     

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.     

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity.     

Kinetics and mechanisms of the recombination of Zn2+, Co2+, and Ni2+ with the metal-depleted catalytic site of horse liver alcohol dehydrogenase

The kinetics of the recombination of the metal-depleted active site of horse liver alcohol dehydrogenase (LADH) with metal ions have been studied over a range of pH and temperature. The formation rates were determined optically, by activity measurements, or by using the pH change during metal incorporation with a pH-indicator as monitor. The binding of Zn2+, Co2+, and Ni2+ ions occurs in a two-step process. The first step is a fast equilibrium reaction, characterized by an equilibrium constant K1. The spectroscopic and catalytic properties of the native or metal-substituted protein are recovered in a slow, monomolecular process with the rate constant k2. The rate constants k2 5.2 X 10(-2) sec-1 (Zn2+), 1.1 X 10(-3) sec-1 (Co2+), and 2 X 10(-4) sec-1 (Ni2+). The rate constants increase with increasing pH. Using temperature dependence, the activation parameters for the reaction with Co2+ and Ni2+ were determined. Activation energies of 51 +/- 2.5 kJ/mol (0.033 M N-Tris-(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES), pH 6, 9) for Co2+ and 48.5 +/- 4 kJ/mol (0.033 M TES, pH 7, 2) for Ni2+ at 23 degrees C were found. The correspondent activation entropies are - 146 +/- 10 kJ/mol K for Co2+ and - 163 +/- 9 kJ/mol K for Ni2+. Two protons are released during the binding of Zn2+ to H4Zn(n)2 LADH in the pH range 6.8-8.1. The binding of coenzyme, either reduced or oxidized, prevents completely the incorporation of metal ions, suggesting that the metal ions enter the catalytic site via the coenzyme binding domain and not through the hydrophobic substrate channel.     

Carboxypeptidase A: mechanism of zinc inhibition

Zinc ions competitively inhibit carboxypeptidase A from bovine pancreas. The state(s) of hydroxylation of zinc and their possible site(s) of interaction with the enzyme have been investigated by determining the strength of zinc inhibition over pH range 4.6-10.5. The inhibition kinetics were recorded under stopped-flow conditions using the alpha-Val isozyme and the peptide substrate Dns-Gly-Ala-Phe in 0.5 M NaCl at 25 degrees C. The pH dependence of pKI follows a pattern which indicates that the enzyme is selectively inhibited by zinc monohydroxide, ZnOH+ (KI = 7.1 X 10(-7) M). The formation of the inhibitory ZnOH+ complex from fully hydrated Zn2+ is characterized by an ionization constant of 9.05, and the consecutive conversion of ZnOH+ to Zn(OH)2, Zn(OH)3-, and Zn(OH)4(2-) complexes takes place with ionization constants of 9.75, 10.1, and 10.5, respectively. Ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for binding of the ZnOH+ complex. The enzymatic activity (kcat/Km) is influenced by three ionizable groups: pKEH2 5.78, pKEH 8.60, and pKE 10.2. Since the values of pKLH and pKEH2 are virtually identical, it is possible that the inhibitory ZnOH+ complex interacts with the group responsible for pKEH2. Previous studies have suggested that pKEH2 reflects the ionization of Glu-270 and its interaction with a water molecule coordinated to the catalytic zinc ion. It is proposed that the inhibitory zinc ion binds to the carboxylate of Glu-270 and that the inhibition process is specific for zinc monohydroxide because it allows the formation of a stabilizing hydroxide bridge between the inhibitory and catalytic zinc ions.     

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.     

GTP cyclohydrolase I utilizes metal-free GTP as its substrate.

GTP cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin and its activity is important in the regulation of monoamine neurotransmitters such as dopamine, norepinephrine and serotonin. We have studied the action of divalent cations on the enzyme activity of purified recombinant human GCH expressed in Escherichia coli. First, we showed that the enzyme activity is dependent on the concentration of Mg-free GTP. Inhibition of the enzyme activity by Mg2+, as well as by Mn2+, Co2+ or Zn2+, was due to the reduction of the availability of metal-free GTP substrate for the enzyme, when a divalent cation was present at a relatively high concentration with respect to GTP. We next examined the requirement of Zn2+ for enzyme activity by the use of a protein refolding assay, because the recombinant enzyme contained approximately one zinc atom per subunit of the decameric protein. Only when Zn2+ was present was the activity of the denatured enzyme effectively recovered by incubation with a chaperone protein. These are the first data demonstrating that GCH recognizes Mg-free GTP and requires Zn2+ for its catalytic activity. We suggest that the cellular concentration of divalent cations can modulate GCH activity, and thus tetrahydrobiopterin biosynthesis as well.     

Is erythrocyte alkaline phosphatase activity a marker of zinc status in humans?

The identification of an enzyme activity that responds to changes in Zn intake may serve as a useful biomarker for Zn status. Alkaline phosphatase (ALP) is a dimeric protein with each subunit containing two Zn atoms. The activity of ALP in erythrocytes (E) decreases as a result of a low Zn diet, which suggests that this enzyme may be a marker of Zn status. To investigate this further, we determined the response of E-ALP in six healthy subjects following supplementation with 50 mg Zn (4.2×RDI) daily for 4 wk. A small but significant increase in plasma Zn was observed with supplementation (p<0.05), whereas there was no significant change in E-Zn over the same period. Plasma and E-Cu showed no change. Conversely, the activity of E-ALP increased in all subjects from 1.7±0.5 to 5.9±0.7 U/g protein (mean±SE) (p<0.0001). The small change observed in plasma Zn is not biologically significant in view of the many documented factors that influence its concentration. Our data support the hypothesis that E-ALP is a marker of Zn status in humans.     

Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.     

Crystal structures of nonoxidative zinc-dependent 2,6-dihydroxybenzoate (gamma-resorcylate) decarboxylase from Rhizobium sp. strain MTP-10005

Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one (alphabeta)8 triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu8, His10, His164, Asp287, and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp287, which forms a triad together with His218 and Glu221 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.     

Activation of sphingomyelinase from Bacillus cereus by Zn2+ hitherto accepted as a strong inhibitor

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg2+, Mn2+, and Co2+, but strongly inhibited by Zn2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn2+ and that the Zn2+ binding to the high-affinity site can activate the enzyme, whereas the Zn2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn2+ binding to the high-affinity site, but was competitively inhibited by the Zn2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg2+ = Co2+ < Mn2+ < Zn2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.     

Activity and properties of fructose bisphosphatase of turbellarian Phagocata sibirica

Activity and properties of fructose bisphosphatase (FBPase) was studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. There was studied dependence of the FBPase reaction rate on the substrate concentration. For realization of the enzyme activity, the high affinity to substrate and presence of bivalent cations (Mg2+ or Mn2+) are necessary. The was studied the effect of various effectors as well as of monovalent (Na+, K+, Li+, and NH4+) and bivalent (Zn2+ and Cu2+) cations.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex.

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.     

Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.