MetAP2 preferentially removes iMet from the N terminus of β-globin, whereas MetAP1 is inactive. (A) MetAP2 is the eukaryotic MetAP that is competent to remove iMet from the N-terminal valine found on both globins as the unfolded globin peptide emerges from the ribosome. Inhibition of MetAP2 prevents iMet removal, allowing N-terminal acetylation by the catalytic Naa50 subunit of the N-terminal acetyltransferase E complex, retaining iMet and acetyl-iMet on a subset of α-globin and β-globin. (B) Lineweaver-Burk plot showing that MetAP2 removes the N-terminal Met from the β-globin and β^S-globin octapeptides similarly but fivefold more efficiently compared with the rate of removal of Met from the α-globin N-terminal octapeptide. MetAP2 (9.3 nM) was reconstituted in 30 μM Mn²⁺, the physiological divalent metal cofactor.⁵⁴  (C) MetAP1 (57.9 nM) cannot remove Met from the N-terminal β^S-globin peptide, but it efficiently cleaves Met from a standard control Met-Ala-Ser tripeptide substrate. MetAP1 was reconstituted with 30 μM Co²⁺ divalent metal cofactor to maximize activity. MetAP1 and MetAP2 are assayed in a published coupled enzyme assay with L-amino acid oxidase and horseradish peroxidase (Assay 1).⁵⁵  RFU, relative fluorescent unit.