Physiologically Relevant Metal Cofactor for Methionine Aminopeptidase-2 Is Manganese
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- 作者：Jieyi Wang ; George S. Sheppard ; Pingping Lou ; Megumi Kawai ; Chang Park ; David A. Egan ; Andrew Schneider ; Jennifer Bouska ; Rick Lesniewski ; and Jack Henkin
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 6, 2003
- 年：2003
- 卷：42
- 期：17
- 页码：5035 - 5042
- 全文大小：220K
- 年卷期：v.42,no.17(May 6, 2003)
- ISSN：1520-4995
文摘
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 eightdivalent metal ions, including Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+, on recombinanthuman methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptidesubstrates: MAS, MGAQFSKT, and 3H-MASK(biotin)G. The activity of MetAP2 on either MAS orMGAQFSKT was enhanced 15-25-fold by Co2+ or Mn2+ metal ions in a broad concentration range(1-1000 
M). 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 isphysiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selectivefor MetAP2 with different metal cofactors. A-310840 below 10 M did not inhibit the activity of MetAP2-Mn2+ but was very potent against MetAP2 with other metal ions including Co2+, Fe2+, Ni2+, and Zn2+ inthe 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 activityand did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, whileA-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicatingcellular 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 inhibitMetAP2-Mn2+.
M). 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 isphysiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selectivefor MetAP2 with different metal cofactors. A-310840 below 10 M did not inhibit the activity of MetAP2-Mn2+ but was very potent against MetAP2 with other metal ions including Co2+, Fe2+, Ni2+, and Zn2+ inthe 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 activityand did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, whileA-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicatingcellular 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 inhibitMetAP2-Mn2+.