EPR evidence for binuclear manganese(II) centers in rat liver arginase

Reczkowski, Robert S.; Ash, David E.

doi:10.1021/ja00053a064

Cited by 112 publications

(81 citation statements)

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“…The results of the Mn(II) determinations are summarized in Table I. The wild-type enzyme had a full complement of 2 Mn(II)/subunit, in agreement with previously published stoichiometries (2,5). Similar Mn(II) stoichiometries were also determined for the R308A and R308E mutants.…”

Section: Resultssupporting

confidence: 86%

“…The wild-type protein had a K m of 1.1 mM for L-arginine, in good agreement with the values of 1-1.7 mM reported for rat liver arginase (2,5,28,29). The mutant arginases showed K m values for L-arginine that were about 2.5-fold higher compared with wild-type enzyme.…”

Section: Resultssupporting

confidence: 85%

“…Manganese Stoichiometry-Because an intact binuclear Mn(II) center is essential for maximal catalytic activity (2,5), it is possible that the decreased activity of the R308A, R308E, and R308K mutants relative to wild-type enzyme resulted from altered manganese stoichiometries for the mutant enzymes. The Mn(II) stoichiometries for wild-type and the mutant arginases were determined by EPR spectroscopy using samples that were heat-activated in the presence of Mn(II) and then washed exhaustively to remove excess Mn(II).…”

Section: Resultsmentioning

confidence: 99%

“…The physiological activator is Mn(II), and the fully active rat liver enzyme contains 2 Mn(II)/subunit (4,5). The crystal structure of type I arginase from rat liver has been determined at 2.1-Å resolution (4).…”

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confidence: 99%

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Subunit-Subunit Interactions in Trimeric Arginase

Lavulo¹,

Sossong²,

Brigham-Burke³

et al. 2001

Journal of Biological Chemistry

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The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-Å resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554 -557). A key feature of this structure is a novel Sshaped oligomerization motif at the carboxyl terminus of the protein that mediates ϳ54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10 5 . These monomeric arginase variants are catalytically active, with k cat /K m values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6 -65.5°C, while the corresponding value for the wild-type enzyme is 70°C. The three-dimensional structure of the R308K variant has been determined at 3-Å resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.

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Section: Resultssupporting

confidence: 86%

Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Subunit-Subunit Interactions in Trimeric Arginase

Lavulo¹,

Sossong²,

Brigham-Burke³

et al. 2001

Journal of Biological Chemistry

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“…The presence of spinexchange coupling would provide clear evidence that the two Mn(II) ions are forming a binuclear cluster in AntR, as suggested from the MntR structure ( Figure 7). This approach has been used to identify the formation of a binuclear Mn-(II) cluster in aminopeptidases (30,31), concanavalin A (32), phosphatase (33), thiosulfate oxidase (34), arginase (35), phosphotriesterase (36), and enolase (37). As shown in Figure 4, our low-temperature cw EPR spectra lack the spectral signatures associated with spin-exchange coupling and therefore are not consistent with formation of a binuclear manganese cluster in AntR.…”

Section: Discussionmentioning

confidence: 90%

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

et al. 2006

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The anthracis repressor (AntR) is a manganese-activated transcriptional regulator from Bacillus anthracis and is a member of the diphtheria toxin repressor (DtxR) family of proteins. In this paper, we characterize the Mn(II) binding and protein dimerization state using a combination of continuous wave (cw) and pulsed EPR methods. Equilibrium metal binding experiments showed that AntR binds 2 equivalents of Mn(II) with positive cooperativity and apparent dissociation constants of 210 and 16.6 µM. AntR showed sub-millisecond Mn(II) on-rates as measured using stopped-flow EPR. The kinetics of Mn(II) dissociation, measured by displacement with Zn(II), was biphasic with rate constants of 35.7 and 0.115 s -1 . Variable-temperature parallel and perpendicular mode cw EPR spectra showed no evidence of a spin-exchange interaction, suggesting that the two Mn(II) ions are not forming a binuclear cluster. Finally, size exclusion chromatography and double electron-electron resonance EPR demonstrated that AntR forms a dimer in the absence of Mn(II). These results provide insights into the metal activation of AntR and allow a comparison with related DtxR proteins.Bacteria, like all organisms, regulate the amount of transition metal ions in the cell. Of these metals, iron is particularly important as it is essential for viability, but accumulation of iron, especially in the ferrous [Fe(II)] form, results in oxidative damage as the metal is oxidized to the ferric form. Bacteria generally do not accumulate iron or other transition metals as seen in eukaryotic cells and, therefore, require efficient mechanisms for regulating the intracellular concentration of transition metal ions (1). Iron homeostasis is regulated in many bacteria by proteins of the diphtheria toxin repressor (DtxR) 1 family. These proteins function as metal ion sensors and regulate the transcription of genes involved in metal uptake, storage, and utilization.DtxR, the prototypical member of this family of repressors, regulates more than 40 genes in Corynebacterium diphtheriae, and IdeR, from Mycobacterium tuberculosis, regulates approximately 45 different genes (2). In many cases, these repressors have been co-opted to regulate the virulence response (1, 3). In C. diphtheriae, toxin gene expression is repressed by the metal-activated form of DtxR when the intracellular Fe(II) levels are above a certain threshold. A drop in the Fe(II) level inactivates the repressor and allows active gene transcription.DtxR proteins typically contain two domains connected by a flexible tether (Figure 1).

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Synthesis and Crystal Structure of Manganese(II) Bipyridine Carboxylato Complexes: [(bipy)2MnII(μ-C2H5CO2)2MnII(bipy)2](ClO4)2 and [MnII(ClCH2CO2)(H2O)(bipy)2]ClO4 · H2O (bipy = 2,2′-bipyridine)

Zhang

Janiak

2001

Z. anorg. allg. Chem.

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Two manganese(II) bipyridine carboxylate complexes, [(bipy) 2 Mn II (l-C 2 H 5 CO 2 ) 2 Mn II (bipy) 2 } 2 ](ClO 4 ) 2 (1), and [Mn II (ClCH 2 CO 2 )(H 2 O)(bipy) 2 ]ClO 4´H2 O (2) were prepared. 1 crystallizes in the triclinic space group P 1 with a = 8.604 (3), b = 12.062(3), c = 13.471(3) A Ê , a = 112.47(2), b = 93.86(2), c = 92.87(3)°, V = 1211.1(6) A Ê 3 and Z = 1. In the dimeric, cationic complex with a crystallographic center of symmetry two 2,2'-bipyridine molecules chelate each manganese atom. These two metal fragments are then bridged by two propionato groups in a syn-anti conformation. The Mn´´´Mn distance is 4.653 A Ê . 2 crystallizes in the mono-clinic space group P2 1 /c with a = 9.042(1), b = 13.891(1), c = 21.022(3) A Ê , b = 102.00(1)°, V = 2569.3(5) A Ê 3 and Z = 4.2 is a monomeric cationic complex in which two bipyridine ligands chelate the manganese atom in a cis fashion. A chloroacetato and an aqua ligand complete the six-coordination. Only in 2 is the intermolecular packing controlled by weak p-stacking besides C±H´´´p contacts between the bipyridine ligands.

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EPR evidence for binuclear manganese(II) centers in rat liver arginase

Cited by 112 publications

References 1 publication

Subunit-Subunit Interactions in Trimeric Arginase

Subunit-Subunit Interactions in Trimeric Arginase

Mn(II) Binding by the Anthracis Repressor from Bacillus anthracis

Synthesis and Crystal Structure of Manganese(II) Bipyridine Carboxylato Complexes: [(bipy)2MnII(μ-C2H5CO2)2MnII(bipy)2](ClO4)2 and [MnII(ClCH2CO2)(H2O)(bipy)2]ClO4 · H2O (bipy = 2,2′-bipyridine)

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