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. Author manuscript; available in PMC: 2023 Jun 9.
Published in final edited form as: Cell. 2022 May 17;185(12):2148–2163.e27. doi: 10.1016/j.cell.2022.04.011

Figure 4. ZNG1 enhances METAP1 aminopeptidase activity in vitro.

Figure 4.

(A) Representative titration of 1.0 μM METAP1, 1.0 μM quin-2 with Zn. (B) Representative titration of 1.3 μM ZNG1, 1.3 μM quin-2 with Zn. Solid lines are global fits of independent replicates; dashed lines are simulations of 10-fold greater and weaker affinities. (C) GTPase activity of ZNG1 with or without Zn and/or Zn2METAP1. (D) Initial rate of Met release from 750 μM MAHAIHY peptide by 20 nM METAP1 in different metalation states. (E) Activation of 10 μM Zn2METAP1 by 25 μM ZNG1 with or without Zn and 500 μM guanine nucleotides. (F) Activation of 10 μM Zn2METAP1 by 22.5 μM Zn or 25 μM ZnZNG1 with or without 500 μM guanine nucleotides, all with 250 μM NTA. In panels E and F, activation reactions were diluted to 20 nM METAP1 prior to monitoring cleavage of 750 μM MAHAIHY. (G) Mechanistic model of GTP hydrolysis-coupled Zn transfer from ZNG1 to the METAP1 active site. See also Figure S4.