Metals in the active site of native protein phosphatase-1

Effects of stably incorporated iron on protein phosphatase‐1 structure and activity

“…These attempts suggest that the incorporation of Zn 2+ during recombinant protein expression in E. coli is more challenging than that of iron and manganese. This is contrary to the situation in mammalian cells, where purification of native PP1 from animal tissue resulted instead in a high incorporation of zinc of 74% (1.48 μ m zinc for a protein concentration of 2 μ m ) with no detectable manganese, and only 8% iron (0.16 μ m iron for a protein concentration of 2 μ m ), which was interpreted as loss of iron during purification and/or storage . When PP1 is expressed in E. coli , incorporation of zinc at a later stage during purification could be more difficult as the enzyme is already folded and the residual divalent cations already bound to the enzyme possibly disturb further zinc incorporation.…”

“…The catalytic subunit of native PP1 contains iron and zinc, and the presence of Mg 2+ cannot be ruled out, whereas Mn 2+ content is almost negligible . Similar to the data on PP1 oxidation , all structural and most biochemical data so far have been obtained using recombinant PP1 containing manganese ions (Mn‐PP1) in the active site .…”

“…Probably due to electrochemical properties resulting in a different nucleophilicity of Fe 2+ compared to Mn 2+ , Fe(II)-PP1a shows in general a lower activity than Mn-PP1a. However, the native enzyme showed a higher activity than Mn-PP1a [8], and one explanation could be that this results from the combination of iron and zinc in the active site of native PP1 exerting an increase in the nucleophilicity of a water molecule and/or electrophilicity of the phosphate moiety in the substrate, or effecting the orientation of the substrate in the cavity [24]. We also found that the relative V/K m was higher with the H3pT3 peptide as a substrate for Fe(II)-PP1a, which together with the inactivity toward pNPP could reflect differences in substrate specificity compared to Mn-PP1a, as observed previously with purified native PP1 N [8].…”

“…The catalytic subunit of native PP1 contains iron and zinc, and the presence of Mg 2+ cannot be ruled out, whereas Mn 2+ content is almost negligible [8]. Similar to the data on PP1 oxidation [7], all structural and most biochemical data so far have been obtained using recombinant PP1 containing manganese ions (Mn-PP1) in the active site [8]. This is due to difficulties in purifying and producing a stable preparation of iron-bound PP1 (Fe-PP1), especially in amounts required for crystallography, which has prevented its use in studies so far [9].…”

The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth

“…Inhibitor-1 and CPI-17) are phosphorylation-dependent or only inhibit a subset of PP1 holoenzymes (Endo et al, 1996;Eto, 2009). Inhibitor-2, Inhibitor-3 and Sds22 have been biochemically identified as inhibitors of PP1, but genetic data from yeast indicate that they are actually positive regulators, possibly involved in the biogenesis of PP1 (Cheng and Chen, 2015;Eiteneuer et al, 2014;Heroes et al, 2015). In contrast, substantial biochemical and cell biological evidence indicates that full-length NIPP1 is a very specific and extremely potent inhibitor of PP1 (Beullens et al, 1992 tested protein substrates of PP1, except for phosphoproteins that are recruited through the FHA domain and probably represent the natural substrates of the PP1-NIPP1 holoenzyme.…”