The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme

The identity of the physiologically relevant metal ions for the methionyl a minopeptidase (MetAP) from Escherichia coli was investigated and is suggest ed to be Fe(II). The metal content of whole cells in the absence and presen ce of expression of the type I MetAP from E. coli was determined by inducti vely coupled plasma (ICP) emission analysis. The observed change in whole c ell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, s ignificant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentra tion being 4.4 and 6.2 times greater than that of manganese and zinc, respe ctively. Activity assays of freshly lysed BL21(DE3) cells containing the pM etAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity. C ontrol experiments with BL21(DE3) without the MetAP plasmid showed no detec table enzymatic activity. Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP fr om E. coli. The MetAP from E. coli as purified was found to be catalyticall y inactive (less than or equal to 2 units/mg). ICP emission analysis of the as-purified enzyme revealed no catalytically relevant metal ions. Both the Co(II)- and Fe(II)-MetAP enzymes are susceptible to autoxidation, so stric t care must be taken to remove all dissolved oxygen. Enzymatic assays perfo rmed under anaerobic conditions indicated that of the di- and trivalent met al cations tested to date, only Co(II) (37.3 units/mg), Fe(II) (29.7 units/ mg), Mn(II) (7.0 units/mg), and Zn(II) (3.3 units/mg) provided detectable l evels of enzymatic activity. In each case, excess metal ions were found to be inhibitory. The observed specific activity of Co(II)MetAP is more than 3 times greater than that previously reported for the MetAP from E. coli [Be n-Bassat, A., et al. (1987) J. Bacteriol. 169, 751-757]. This increase in a ctivity is likely due to the strict exclusion of air from reaction samples. Oxidation of either the Iie(II) or Co(II) form of the enzyme resulted in t he complete loss of catalytic activity. The substrate binding constants (K- m) for Met-Gly-Met-Met binding to the Co(II)- or Fe(TI)-substituted MetAP e nzymes, under anaerobic conditions, were found to be 3.16 and 1.95 mM, resp ectively. The combination of these data suggests that the in vivo metal ion s for the MetAP enzyme from E. coli are likely Fe(II) ions.