Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

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.     

Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.     

Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII

We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.     

Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

The binding mechanism of Mg(2+) at the M3 site of human placental alkaline phosphatase was found to be a slow-binding process with a low binding affinity (K(Mg(app.)) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg(2+)-free and Mg(2+)-containing enzymes by acrylamide showed almost identical dynamic quenching constant (K(sv) = 4.44 +/- 0.09 M(-1)), indicating that there is no gross conformational difference between the M3-free and the M3-Mg(2+) enzymes. However, Zn(2+) was found to have a high affinity with the M3 site (K(Zn(app.)) = 0.11 mM) and was observed as a time-dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (k(obs)) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3'-Zn) having an extremely high affinity for the Zn(2+) (K*(Zn(app.)) = 0.33 microM). The conformation of the M3'-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg(2+) activation and Zn(2+) inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg(2+), has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (K(sv) = 3.86 +/- 0.04 M(-1)), suggesting a conformational change. Both Mg(2+) and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn-inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.     

Metal binding to<Emphasis Type="Italic"> Bacillus subtilis</Emphasis> ferrochelatase and interaction between metal sites

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyses metal insertion into protoporphyrin IX. The location of the metal binding site with respect to the bound porphyrin substrate and the mode of metal binding are of central importance for understanding the mechanism of porphyrin metallation. In this work we demonstrate that Zn(2+), which is commonly used as substrate in assays of the ferrochelatase reaction, and Cd(2+), an inhibitor of the enzyme, bind to the invariant amino acids His183 and Glu264 and water molecules, all located within the porphyrin binding cleft. On the other hand, Mg(2+), which has been shown to bind close to the surface at 7 A from His183, was largely absent from its site. Activity measurements demonstrate that Mg(2+) has a stimulatory effect on the enzyme, lowering K(M) for Zn(2+) from 55 to 24 micro M. Changing one of the Mg(2+) binding residues, Glu272, to serine abolishes the effect of Mg(2+). It is proposed that prior to metal insertion the metal may form a sitting-atop (SAT) complex with the invariant His-Glu couple and the porphyrin. Metal binding to the Mg(2+) site may stimulate metal release from the protein ligands and its insertion into the porphyrin.     

Distinct roles of two Mg2+ binding sites in regulation of murine flap endonuclease-1 activities

Removal of flap DNA intermediates in DNA replication and repair by flap endonuclease-1 (FEN-1) is essential for mammalian genome integrity. Divalent metal ions, Mg(2+) or Mn(2+), are required for the active center of FEN-1 nucleases. However, it remains unclear as to how Mg(2+) stimulates enzymatic activity. In the present study, we systemically characterize the interaction between Mg(2+) and murine FEN-1 (mFEN-1). We demonstrate that Mg(2+) stimulates mFEN-1 activity at physiological levels but inhibits the activity at concentrations higher than 20 mM. Our data suggest that mFEN-1 exists as a metalloenzyme in physiological conditions and that each enzyme molecule binds two Mg(2+) ions. Binding of Mg(2+) to the M1 binding site coordinated by the D86 residue cluster enhances mFEN-1's capability of substrate binding, while binding of the metal to the M2 binding site coordinated by the D181 residue cluster induces conformational changes. Both of these steps are needed for catalysis. Weak, nonspecific Mg(2+) binding is likely responsible for the enzyme inhibition at high concentrations of the cation. Taken together, our results suggest distinct roles for two Mg(2+) binding sites in the regulation of mFEN-1 nuclease activities in a mode different from the "two-metal mechanism".     

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.     

A critical interplay between Ca2+ inhibition and activation by Mg2+ of AC5 revealed by mutants and chimeric constructs

Adenylyl cyclase type 5 (AC5) is sensitive to both high and low affinity inhibition by Ca(2+). This property provides a sensitive feedback mechanism of the Ca(2+) entry that is potentiated by cAMP in sources where AC5 is commonly expressed (e.g. myocardium). Remarkably little is known about the molecular mechanism whereby Ca(2+) inhibits AC5. Because previous studies had showed that Ca(2+) antagonized the activation of adenylyl cyclase brought about by Mg(2+), we have now evaluated the Mg(2+)-binding domain in the catalytic site as the potential site of the interaction, using a number of mutations of AC5 with impaired Mg(2+) activation. Mg(2+) activation exerted contrasting effects on the high and low affinity Ca(2+) inhibition. In both wild type and mutants, activation by Mg(2+) decreased the absolute amount of high affinity inhibition without affecting the K(i) value, whereas the K(i) value for low affinity inhibition was decreased. These effects were directly proportional to the sensitivity of the mutants to Mg(2+). Parallel changes were noted in the efficacies of Ca(2+), Sr(2+), and Ba(2+) in the mutant species, suggesting a simple mutation in a shared domain. Strikingly, forskolin, which activates by a mechanism different from Mg(2+), did not modify inhibition by Ca(2+). Deletion of the N terminus and the C1b domain of AC5 and a chimera formed with AC2 confirmed that the catalytic domain alone was responsible for high affinity inhibition. We therefore conclude that both low and high affinity inhibition by Ca(2+) are exerted on different conformations of the Mg(2+)-binding sites in the catalytic domain of AC5.     

Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.     

Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase.

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.     

Electrocyte (Na(+),K(+))ATPase inhibition induced by zinc is reverted by dithiothreitol

The Mg(2+)-dependent (Na(+),K(+))ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn(2+)) on the (Na(+),K(+))ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn(2+) is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na(+),K(+))ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na(+),K(+))ATPase from both membrane fractions and was able to revert the Zn(2+) inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na(+),K(+))ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na(+),K(+))ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn-dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn(2+) may be considered in regard to its neurotoxic effects.     

The influence of metal ions on malic enzyme activity and lipid synthesis in Aspergillus niger

In the presence of copper significant induction of citric acid overflow was observed, while concomitantly lower levels of total lipids were detected in the cells. Its effect was more obvious in a medium with magnesium as sole divalent metal ions, while in a medium with magnesium and manganese the addition of copper had a less pronounced effect. Since the malic enzyme was recognised as a supplier of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate for lipid biosynthesis, its kinetic parameters with regard to different concentrations of metal ions were investigated. Some inhibition was found with Fe(2+) and Zn(2+), while Cu(2+) ions in a concentration of 0.1 mM completely abolished malic enzyme activity. The same metal ions proportionally reduced the levels of total lipids in Aspergillus niger cells. A strong competitive inhibition of the enzyme by Cu(2+) was observed. It seemed that copper competes with Mg(2+) and Mn(2+) for the same binding site on the protein.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Copper and zinc inhibit Galphas function: a nucleotide-free state of Galphas induced by Cu2+ and Zn2+

The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu(2+) and Zn(2+) inhibit the steady-state GTPase activity of the alpha subunit of GTP-binding protein (Galpha(s)) but do not alter its intrinsic GTPase activity. Cu(2+) and Zn(2+) decrease steady-state GTPase activity by inhibiting the binding of GTP to Galpha(s). Moreover, Cu(2+) and Zn(2+) increase GDP dissociation from Galpha(s) and render the G protein in a nucleotide-free state. However, these cations do not alter the dissociation of the guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) that is already bound to the Galpha(s). Because of their ability to inhibit GTPgammaS binding, preincubation of Cu(2+) or Zn(2+) with Galpha(s) does not permit GTPgammaS to activate Galpha(s) and stimulate AC activity. However, preincubation of Galpha(s) with GTPgammaS followed by addition of Cu(2+) or Zn(2+) did not alter the ability of Galpha(s) to stimulate AC activity. Interestingly, AlF(4)(-) partially restored the ability of Galpha(s), which had been preincubated with Cu(2+) or Zn(2+), to stimulate AC; AlF(4)(-) does not permit the re-association of unbound GDP with Galpha(s). Thus, the interaction of AlF(4)(-) with the nucleotide-free Galpha(s) is sufficient to activate AC. Using antibodies to the N and C termini of Galpha(s), we show that the Cu(2+) interaction site on the G protein is in the C terminus. We conclude that Cu(2+) and Zn(2+) generate a nucleotide-free state of Galpha(s) and that, in the absence of any nucleotide, the gamma-phosphate mimic of GTP, AlF(4)(-), alters Galpha(s) structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu(2+) and Zn(2+) as compared with forskolin-stimulated activity is consistent with Galpha(s) being a primary target of these cations in regulating the signaling from receptor to AC.     

Modulation of cyclin-dependent kinase 4 by binding of magnesium (II) and manganese (II)

All kinases require an essential divalent metal for their activity. In this study, we investigated the metal dependence of cyclin-dependent kinase 4 (CDK4). With Mg(2+) as the essential metal and MgATP being the variable substrate, the maximum velocity, V, was not affected by changes in metal concentration, whereas V/K was perturbed, indicating that the metal effects were mainly derived from a change in the K(m) for MgATP. Analysis of the metal dependence of initial rates according to a simple metal binding model indicated the presence on enzyme of one activating metal-binding site with a dissociation constant, K(d(a)), of 5 +/-1 mM, and three inhibitory metal-binding sites with an averaged dissociation constant, K(d(i)), of 12+/-1 mM and that the binding of metal to the activating and inhibitory sites appeared to be ordered with binding of metal to the activating site first. Substitution of Mn(2+) for Mg(2+) yielded similar metal dependence kinetics with a value of 1.0+/-0.1 and 4.7+/-0.1 for K(d(a)) and K(d(i)), respectively. The inhibition constants for the inhibition of CDK4 by MgADP and a small molecule inhibitor were also perturbed by Mg(2+). K(d(a)) values estimated from the metal variation of the inhibition of CDK4 by MgADP (6+/-3 mM) and a small molecule inhibitor (3+/-1 mM), were in good agreement with the K(d(a)) value (5+/-1 mM) obtained from the metal variation of the initial rate of CDK4. By using the van't Hoff plot, the temperature dependence of K(d(a)) and K(d(i)) yielded an enthalpy of -6.0 +/- 1.1 kcal/mol for binding of Mg(2+) to the activating site and -3.2 +/- 0.6 kcal/mol for Mg(2+) binding to the inhibitory sites. The values of associated entropy were also negative, indicating that these metal binding reactions were entirely enthalpy-driven. These data were consistent with metal binding to multiple sites on CDK4 that perturbs the enzyme structure, modulates the enzyme activity, and alters the affinities of inhibitor for the metal-bound enzyme species. However, the affinities of small molecule inhibitors for CDK4 were not affected by the change of metal from Mg(2+) to Mn(2+), suggesting that the structures of enzyme-Mg(2+) and enzyme-Mn(2+) were similar.     

Metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase

Bovine lens leucyl aminopeptidase (blLAP), a homohexameric metallopeptidase preferring bulky and hydrophobic amino acids at the N-terminus of (di)peptides, contains two Zn(2+) ions per subunit that are essential for catalytic activity. They may be replaced by other divalent cations with different exchange kinetics. The protein readily exchangeable site (site 1) can be occupied by Zn(2+), Mn(2+), Mg(2+), or Co(2+), while the tight binding site (site 2) can be occupied by Zn(2+) or Co(2+). We recently reported that introduction of Mn(2+) into site 1 generates a novel activity of blLAP toward CysGly [Cappiello, M., et al. (2004) Biochem. J. 378, 35-44], which in contrast is not hydrolyzed by the (Zn/Zn) enzyme. This finding, while disclosing a potential specific role for blLAP in glutathione metabolism, raised a question about the features required for molecules to be a substrate for the enzyme. To clarify the interaction of the enzyme with sulfhydryl-containing derivatives, (Zn/Zn)- and (Mn/Zn)blLAP forms were prepared and functional-structural studies were undertaken. Thus, a kinetic analysis of various compounds with both enzyme forms was performed; the crystal structure of (Zn/Zn)blLAP in complex with the peptidomimetic derivative Zofenoprilat was determined, and a modeling study on the CysGly-(Zn/Zn)blLAP complex was carried out. This combined approach provided insight into the interaction of blLAP with sulfhydryl-containing derivatives, showing that the metal exchange in site 1 modulates binding to these molecules that may result in enzyme substrates or inhibitors, depending on the nature of the metal.     

Crystal structures of fructose 1,6-bisphosphatase: mechanism of catalysis and allosteric inhibition revealed in product complexes

Crystal structures of metal-product complexes of fructose 1, 6-bisphosphatase (FBPase) reveal competition between AMP and divalent cations. In the presence of AMP, the Zn(2+)-product and Mg(2+)-product complexes have a divalent cation present only at one of three metal binding sites (site 1). The enzyme is in the T-state conformation with a disordered loop of residues 52-72 (loop 52-72). In the absence of AMP, the enzyme crystallizes in the R-state conformation, with loop 52-72 associated with the active site. In structures without AMP, three metal-binding sites are occupied by Zn(2+) and two of three metal sites (sites 1 and 2) by Mg(2+). Evidently, the association of AMP with FBPase disorders loop 52-72, the consequence of which is the release of cations from two of three metal binding sites. In the Mg(2+) complexes (but not the Zn(2+) complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site 1 and is oriented for a nucleophilic attack on the bound phosphate molecule. A mechanism is presented for the forward reaction, in which Asp74 and Glu98 together generate a hydroxide anion coordinated to the Mg(2+) at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg(2+) at site 1.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.