Document Type

Article

Language

eng

Format of Original

12 p.

Publication Date

12-2008

Publisher

FEBS

Source Publication

FEBS Journal

Source ISSN

1742-646X

Original Item ID

DOI: 10.1111/j.1742-4658.2008.06749.x, PubMed Central: PMC2699115

Abstract

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) dd 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.

Comments

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

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