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Cooperative activation of human mitochondrial Methionine Aminopeptidase (hmMetAP) by Co2+
Author(s) -
Hu Xiaoyi,
Mildvan Albert Samuel,
Liu Jun O
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a272-b
Subject(s) - methionine , chemistry , enzyme kinetics , substrate (aquarium) , catalysis , stereochemistry , metal ions in aqueous solution , recombinant dna , amino acid , divalent , metal , active site , biochemistry , biology , ecology , organic chemistry , gene
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 Co 2+ , much more than by Zn 2+ , with values of k cat = 0.37 ± 0.02 min −1 and K m = 0.59 ± 0.07 mM at pH 8.0 and 25°C. At saturating substrate, a plot of V max versus free Co 2+ shows sigmoidal metal activation, both with and without the His‐tag, with a Hill coefficient of 1.6 ± 0.2 and a K 0.5 of 0.5 ± 0.1 μM. Hence, at least two Co 2+ ions act cooperatively to promote catalysis, providing kinetic evidence for the functioning of both metal ions of the binuclear complex. This research was supported by NIH Grant CA078743.