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Analyzing the Catalytic Role of Asp97 in the Methionine Aminopeptidase from Escherichia coli
FEBS Journal
  • Sanghamitra Mitra, Utah State University
  • Kathleen M. Job, Utah State University
  • Lu Meng, Utah State University
  • Brian Bennett, Marquette University
  • Richard C. Holz, Marquette University
Document Type
Format of Original
12 p.
Publication Date
Original Item ID
DOI: 10.1111/j.1742-4658.2008.06749.x, PubMed Central: PMC2699115
An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co(II) d–d spectra for wild-type, D97E and D97A EcMetAP-I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) EcMetAP-I, and only a doubling of intensity was observed upon addition of a second Co(II) ion. In contrast, the electronic absorption spectra of [Co_(D97N EcMetAP-I)] and [CoCo(D97N EcMetAP-I)] were distinct, as were the EPR spectra. On the basis of the observed molar absorptivities, the Co(II) ions binding to the D97E, D97A and D97N EcMetAP-I active sites are pentacoordinate. Combination of these data suggests that mutating the only nonbridging ligand in the second divalent metal-binding site in MetAPs to an alanine, which effectively removes the ability of the enzyme to form a dinuclear site, provides a MetAP enzyme that retains catalytic activity, albeit at extremely low levels. Although mononuclear MetAPs are active, the physiologically relevant form of the enzyme is probably dinuclear, given that the majority of the data reported to date are consistent with weak cooperative binding.

Accepted version. FEBS Journal, Vol. 275, No. 24 (December 2008): 6248-6259. DOI. © 2008 FEBS. Used with permission.

This is the peer reviewed version of the following article: FEBS Journal, Vol. 275, No. 24 (December 2008): 6248-6259, which has been published in final form at DOI. This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

Richard C. Holz was affiliated with Loyola University-Chicago at the time of publication.

Citation Information
Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, et al.. "Analyzing the Catalytic Role of Asp97 in the Methionine Aminopeptidase from Escherichia coli" FEBS Journal (2008) ISSN: 1742-646X
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