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Kinetic and Spectroscopic Characterization of the D97 altered Methionine aminopeptidase from Escherichia coli
Author(s) -
Mitra Sanghamitra,
Job Kathleen M.,
Bennett Brian,
Holz Richard C.
Publication year - 2006
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.20.5.lb53-b
Subject(s) - methionine , residue (chemistry) , chemistry , enzyme , aminopeptidase , stereochemistry , histidine , escherichia coli , carboxylate , active site , biochemistry , amino acid , leucine , gene
The removal of N‐terminal methionine from proteins and polypeptides is dependent upon a novel class of protease metalloenzyme methionine aminopeptidase (MetAP). MetAPs are highly specific for N‐terminal methionine residues and their enzymatic activities are restricted to substrates that have physically small and uncharged amino acid residue adjacent to the N‐terminal methionine. All the MetAPs that have been crystallographically characterized to date have identical catalytic domains that contain a bis(μ‐carboxylato)(μ‐aquo/hydroxo)dicobalt core with an additional carboxylate residue at each metal site and a single histidine residue bound to one of the metal ions. D97 is a bidentate ligand to the second metal ion. In humans, in a MetAP related enzyme this residue is found to be altered in some abnormalities of the skin and causes mental retardation. To gain insight into the role of the strictly conserved aspartate residue, D97, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli ( Ec MetAP‐I), D97A, D97E and D97N altered enzymes were prepared. These variants have been both kinetically and spectroscopically characterized. The results strongly suggest that not only the aspartate residue is catalytically important but also is an essential residue for positioning the substrate to form the transition state. This work was supported by NSF (CHE‐0549221).