The Methionyl Aminopeptidase from Escherichia coli Can Function as an Iron(II) Enzyme

• Published in: Biochemistry (Easton) Vol. 38; no. 34; pp. 11079 - 11085
• Main Authors: D'souz, Ventris M, Holz, Richard C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24.08.1999
• Subjects: Aminopeptidases - biosynthesis; Aminopeptidases - metabolism; Anaerobiosis; Binding Sites; Cations, Divalent; Cobalt - chemistry; Cobalt - metabolism; Enzyme Activation; Enzyme Induction; Escherichia coli; Escherichia coli - enzymology; Ferric Compounds - chemistry; Ferric Compounds - metabolism; Humans; Kinetics; Metals, Heavy - chemistry; Metals, Heavy - metabolism; Methionyl Aminopeptidases; Peptides - chemistry; Peptides - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990872h

The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II). The metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli was determined by inductively coupled plasma (ICP) emission analysis. The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentration being 4.4 and 6.2 times greater than that of manganese and zinc, respectively. Activity assays of freshly lysed BL21(DE3) cells containing the pMetAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity. Control experiments with BL21(DE3) without the MetAP plasmid showed no detectable enzymatic activity. Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP from E. coli. The MetAP from E. coli as purified was found to be catalytically inactive (≤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 strict care must be taken to remove all dissolved oxygen. Enzymatic assays performed under anaerobic conditions indicated that of the di- and trivalent metal 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 levels 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 [Ben-Bassat, A., et al. (1987) J. Bacteriol. 169, 751−757]. This increase in activity is likely due to the strict exclusion of air from reaction samples. Oxidation of either the Fe(II) or Co(II) form of the enzyme resulted in the complete loss of catalytic activity. The substrate binding constants (K m) for Met-Gly-Met-Met binding to the Co(II)- or Fe(II)-substituted MetAP enzymes, under anaerobic conditions, were found to be 3.16 and 1.95 mM, respectively. The combination of these data suggests that the in vivo metal ions for the MetAP enzyme from E. coli are likely Fe(II) ions.