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Cooperative activation of human mitochondrial Methionine Aminopeptidase (hmMetAP) by Co2+

Methionine Aminopeptidases (MetAPs) proteolytically remove N‐terminal methionines from proteins. X‐ray structures of MetAPs show two divalent cations at the active site which share Asp, Glu, and aquo ligands (Biochem. 44, (2005)). Whether one or both metal ions of this binuclear complex function in catalysis has been a subject of controversy. The recombinant hmMetAP, a type Id MetAP, was cloned and prepared under metal‐free conditions, both with and without an amino‐terminal His‐tag. With Met‐Pro‐pNitroanalide as substrate, the inactive MetAP was readily activated by Co2+, much more than by Zn2+, with values of kcat = 0.37 ± 0.02 min−1 and Km = 0.59 ± 0.07 mM at pH 8.0 and 25°C. At saturating substrate, a plot of Vmax versus free Co2+ shows sigmoidal metal activation, both with and without the His‐tag, with a Hill coefficient of 1.6 ± 0.2 and a K0.5 of 0.5 ± 0.1 μM. Hence, at least two Co2+ ions act cooperatively to promote catalysis, providing kinetic evidence for the functioning of both metal ions of the binuclear complex.