The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+, on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and 3H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co2+ or Mn2+ metal ions in a broad concentration range (1-1000 μM). In the presence of reduced glutathione to mimic the cellular environment, Co2+ and Mn2+ were also the best stimulators (∼30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 μM did not inhibit the activity of MetAP2Mn2+ but was very potent against MetAP2 with other metal ions including Co2+, Fe2+, Ni2+, and Zn2+ in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn2+, as well as Co2+, Fe2+, Ni2+, and Zn2+. In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn2+.
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