Analysis of the stoichiometric metal activation of methionine aminopeptidase.
BACKGROUND
Methionine aminopeptidase (MetAP) is a ubiquitous enzyme required for cell survival and an attractive target for antibacterial and anticancer drug development. The number of a divalent metal required for catalysis is under intense debate. E. coli MetAP was shown to be fully active with one equivalent of metal by graphical analysis, but it was inferred to require at least two metals by a Hill equation model. Herein, we report a mathematical model and detailed analysis of the stoichiometric activation of MetAP by metal cofactors.


RESULTS
Because of diverging results with significant implications in drug discovery, the experimental titration curve for Co2+ activating MetAP was analyzed by fitting with a multiple independent binding sites (MIBS) model, and the quality of the fitting was compared to that of the Hill equation. The fitting by the MIBS model was clearly superior and indicated that complete activity is observed at a one metal to one protein ratio. The shape of the titration curve was also examined for activation of metalloenzymes in general by one or two metals.


CONCLUSIONS
Considering different scenarios of MetAP activation by one or two metal ions, it is concluded that E. coli MetAP is fully active as a monometalated enzyme. Our approach can be of value in proper determination of the number of cations needed for catalysis by metalloenzymes.
Chai
Sergio C
SC
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA. schai@iupui.edu
Ye
Qi-Zhuang
QZ
D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D008958:Models, Molecular
D008670:Metals; D003035:Cobalt; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases