A Mn(II)–Mn(II) center in human prolidase

Human prolidase, the enzyme responsible for the hydrolysis of the Xaa-Pro/Hyp peptide bonds, is a key player in the recycling of imino acids during the final stage of protein catabolism and extracellular matrix remodeling. Its metal active site composition corresponding to the maximal catalytic activity is still unknown, although prolidase function is of increasing interest due to the link with carcinogenesis and mutations in prolidase gene cause a severe connective tissue disorder. Here, using EPR and ICP-MS on human recombinant prolidase produced in Escherichia coli (hRecProl), the Mn(II) ion organized in a dinuclear Mn(II)–Mn(II) center was identified as the protein cofactor. Furthermore, thermal denaturation, CD/fluorescence spectroscopy and limited proteolysis revealed that the Mn(II) is required for the proper protein folding and that a protein conformational modification is needed in the transition from apo- to Mn(II)loaded-enzyme. The collected data provided a better knowledge of the human holo-prolidase and, although limited to the recombinant enzyme, the exact identity and organization of the metal cofactor as well as the conformational change required for activity were proven.     

Yeast glyoxalase I is a monomeric enzyme with two active sites

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.     

Preparation and characterization of various Escherichia coli RNA polymerases containing one or two intrinsic metal ions

The Escherichia coli DNA-dependent RNA polymerase (RPase) holoenzyme (alpha 2 beta beta' sigma) possesses 2 mol equiv of Zn: beta and beta' subunits each contain one Zn ion. An in vitro metal-substitution method developed earlier (method I) was used to remove the two intrinsic Zn ions and then to reconstitute other metal ions into the beta subunit of RPase. One Cd or Hg ion was successfully reconstituted into half-active enzymes (rec-Cd1- or rec-Hg1-RPase), while Mn or Ni ion was not incorporated. A new, simplified in vitro metal-substitution method (method II), which omitted the low-pH treatment and subsequent urea dialysis in method I, was devised in this study. Consequently, Zn or Cd could be incorporated into both the beta and beta' subunits, resulting in rec-Zn2- or rec-Cd2-RPase, respectively. However, only one Hg was incorporated, probably due to steric hindrance by the large size of the Hg ion, while Mn, Ni, or Cr was not bound by the reconstituted enzyme, which instead incorporated only one Zn. Analysis of the metal content of various reconstituted RPases indicated that without low-pH treatment Zn bound to both the beta and beta' subunits when Zn concentrations were higher than 2 X 10(-6)M, but it bound only to the beta' subunit at lower concentrations. Moreover, low-pH treatment destroys the metal binding site in the beta' subunit. The metal sites on the beta and beta' subunits did not have significant affinity for the transition metals such as Mn, Ni, and Cr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defining metal ion inhibitor interactions with recombinant human H- and L-chain ferritins and site-directed variants: an isothermal titration calorimetry study

Zinc and terbium, inhibitors of iron incorporation in the ferritins, have been used for many years as probes of structure-function relationships in these proteins. Isothermal titration calorimetric and kinetic measurements of Zn(II) and Tb(III) binding and inhibition of Fe(II) oxidation were used to identify and characterize thermodynamically ( n, K, Delta H degrees, Delta S degrees, and Delta G degrees ) the functionally important binding sites for these metal ions in recombinant human H-chain, L-chain, and H-chain site-directed variant ferritins. The data reveal at least two classes of binding sites for both Zn(II) and Tb(III) in human H-chain ferritin: one strong, corresponding to binding of one metal ion in each of the eight three-fold channels, and the other weak, involving binding at the ferroxidase and nucleation sites of the protein as well as at other weak unidentified binding sites. Zn(II) and Tb(III) binding to recombinant L-chain ferritin showed similar stoichiometries for the strong binding sites within the channels, but fewer weaker binding sites when compared to the H-chain protein. The kinetics and binding data indicate that the binding of Zn(II) and Tb(III) in the three-fold channels, which is the main pathway of iron(II) entry in ferritin, blocks the access of most of the iron to the ferroxidase sites on the interior of the protein, accounting for the strong inhibition by these metal ions of the oxidative deposition of iron in ferritin.     

Self-assembly of the binuclear metal center of phosphotriesterase

The active site of the bacterial phosphotriesterase (PTE) from Pseudomonas diminuta contains two divalent metal ions and a carboxylated lysine residue. The native enzyme contains two Zn(2+) ions, which can be replaced with Co(2+), Cd(2+), Ni(2+), or Mn(2+) without loss of catalytic activity. Carbon dioxide reacts with the side chain of lysine-169 to form a carbamate functional group within the active site, which then serves as a bridging ligand to the two metal ions. The activation of apo-PTE using variable concentrations of divalent metal ions and bicarbonate was measured in order to establish the mechanism by which the active site of PTE is self-assembled. The time courses for the activation of apo-PTE are pseudo-first-order, and the observed rate constants are directly proportional to the concentration of bicarbonate. In contrast, the apparent rate constants for the activation of apo-PTE decrease as the concentrations of the divalent cations are increased and then become constant at higher concentrations of the divalent metal ions. These results are consistent with a largely ordered kinetic mechanism for the assembly of the binuclear metal center where CO(2)/bicarbonate reacts with the apo-PTE prior to the binding of the two metal ions. When apo-PTE is titrated with 0-8 equiv of Co(2+), Cd(2+), or Zn(2+), the concentration of activated enzyme increases linearly until 2 equiv of metal ion is added and then remains constant at elevated levels of the divalent cations. These results are consistent with the synergistic binding of the two metal ions to the active site, and thus the second metal ion binds more tightly to the protein than does the first metal ion. Measurement of the mean dissociation constant indicates that metal binding to the binuclear metal center is strong [(K(alpha)K(beta))(1/2) = 6.0 x 10(-)(11) M and k(off) = 1.5 x 10(-)(3) min(-)(1) for Zn(2+)]. The removal of the carbamate bridge through the mutagenesis of Lys-169 demonstrates that the carbamate bridge is required for both efficient catalysis and overall stability of the metal center.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献   

Structure of the bacteriophage lambda Ser/Thr protein phosphatase with sulfate ion bound in two coordination modes

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.     

Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions

H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease-ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn(2+)-bound and Mn(2+)-bound complexes in the presence of phosphate ions at resolutions of 2.6 A to 2.0 A. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel beta-strands linked to a C-terminal alpha-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg(2+)-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn(2+) or Mn(2+) ions were observed in the center of the H-N-H motif in cases of Zn(2+) or Mn(2+)-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.     

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.     

Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase

The crystal structure of the tetrameric alpha2beta2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 A resolution. Surprisingly, the two alpha subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the alpha subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.     

Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.     

Structure of the prolidase from Pyrococcus furiosus

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.     

Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels

Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.     

Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism

The number of metal ions required for phosphoryl transfer in restriction endonucleases is still an unresolved question in molecular biology. The two Ca(2+) and Mn(2+) ions observed in the pre- and post-reactive complexes of BamHI conform to the classical two-metal ion choreography. We probed the Mg(2+) cofactor positions at the active site of BamHI by molecular dynamics simulations with one and two metal ions present and identified several catalytically relevant sites. These can mark the pathway of a single ion during catalysis, suggesting its critical role, while a regulatory function is proposed for a possible second ion.     

An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction

The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.     

Isolation of the leucine aminopeptidase gene from Aeromonas proteolytica. Evidence for an enzyme precursor.

The leucine aminopeptidase of Aeromonas proteolytica (EC 3.4.11.10) is a monomeric metalloenzyme having the capacity to bind two Zn2+ atoms in the active site. Structural information of this relatively small aminopeptidase that could illuminate the catalytic mechanism of the metal ions is lacking; hence, we have obtained sequences from the purified enzyme, cloned the corresponding gene, and expressed the recombinant protein in Escherichia coli. The deduced primary amino acid sequence of this secreted protease suggests a potential signal peptide at the NH2 terminus. Expression of the recombinant and native proteins in E. coli and in extracts of culture media of A. proteolytica indicates that the aminopeptidase is secreted as an active and thermosensitive 43-kDa protein that is rapidly transformed to thermostable forms of 30 and 32 kDa. Comparison of the deduced amino acid sequence of the A. proteolytica leucine aminopeptidase with other Zn(2+)-binding metalloenzymes failed to show homologies to the consensus binding sequence His-Glu-X-X-His for the metal ion.