Arginase from Saccharomyces cerevisiae has long been known to be a metal ion-requiring enzyme as it requires heating at 45 degrees C in the presence of 10 mM Mn2+ for catalytic activation. Metals are also thought to play a structural role in the enzyme, but the identity of the structural metal and its precise structural role have not been defined. Analysis of the metal ions that bind to yeast arginase by atomic absorption spectroscopy reveals that there is a weakly associated Mn2+ that binds to the trimeric enzyme with a stoichiometry of 1.04 +/- 0.05 mol of Mn2+ bound per subunit and an apparent K'D value of 26 microM at pH 7.0 and 4 degrees C. A more tightly associated Zn2+ ion can only be removed by dialysis against chelating agents. In occasional preparations, this site contained some Mn2+; however, Zn2+ and Mn2+ together bind to high affinity sites with a stoichiometry of 1.14 +/- 0.25/mol of subunit. Both the loosely associated catalytic Mn2+ ion and the more tightly associated structural Zn2+ ion confer stability to the enzyme. Removal of the weakly bound Mn2+ ion results in a 3 degree C decrease in the midpoint of the thermal transition (T 1/2) (from 57 by 54 degrees C) as monitored by UV difference absorption spectroscopy. Removal of the tightly bound Zn2+ ion produces a 19 degrees C decrease in T 1/2 (to 38 degrees C). Similar results are obtained by circular dichroism measurements. When the Zn2+ ion is removed, the steady-state fluorescence intensity increases 100% as compared to the holoenzyme, with a shift in the emission maximum from 337 to 352 nm. This suggests that in the folded trimeric metalloenzyme, the tryptophan fluorescence is quenched and that upon removal of the structural metal, the quenching is relieved as tryptophan residues become exposed to more polar environments. Equilibrium sedimentation experiments performed after dialysis of the enzyme against EDTA demonstrate that arginase exists in a reversible monomer-trimer equilibrium, in the absence of metal ions, with a KD value of 5.05 x 10(-11) M2. In contrast, the native enzyme exists as a trimer with no evidence of dissociation when Mn2+ and Zn2+ are present (Eisenstein, E., Duong, L.T., Ornberg, R. L., Osborne, J.C., Jr., and Hensley, P. (1986) J. Biol. Chem. 261, 12814-12819). In summary, the study presented here demonstrates that binding of a weakly bound Mn2+ ion confers catalytic activity. In contrast, binding of a more tightly associated Zn2+ ion confers substantial stability to the tertiary and quaternary structure of the enzyme.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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