Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)]2 cluster.
The interaction of bacteriophage lambda protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage lambda protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases.
Rusnak
F
F
Department of Biochemistry and Molecular Biology, Section of Hematology Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA. rusnak@mayo.edu
Yu
L
L
Todorovic
S
S
Mertz
P
P
D010582:Bacteriophage lambda / Q000201:enzymology*; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism*; D017895:Manganese Compounds / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017186:Titrimetry
D002413:Cations, Divalent; D002712:Chlorides; D017895:Manganese Compounds; D011994:Recombinant Proteins; D008345:Manganese; D010749:Phosphoprotein Phosphatases; C025340:manganese chloride