Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

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⁺.