The purification and characterization of arginase from Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007.
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Green
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Eisenstein
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McPhie
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Hensley
D001119:Arginase / Q000302:isolation & purification* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D007700:Kinetics; D008345:Manganese / Q000032:analysis; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002614:Chelating Agents; D008345:Manganese; D001119:Arginase