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Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli
Author(s) -
Mitra Sanghamitra,
Job Kathleen M.,
Meng Lu,
Bennett Brian,
Holz Richard C.
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06749.x
Subject(s) - chemistry , alanine , stereochemistry , active site , asparagine , binding site , enzyme , serine , amino acid , biochemistry
An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli ( Ec MetAP‐I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal‐binding site in Ec MetAP‐I. These mutant Ec MetAP‐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 Ec MetAP‐Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000‐fold decrease in k ca towards Met‐Gly‐Met‐Met as compared to the wild‐type enzyme. The Co(II) d – d spectra for wild‐type, D97E and D97A Ec MetAP‐I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) Ec MetAP‐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 Ec MetAP‐I)] and [CoCo(D97N Ec MetAP‐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 Ec MetAP‐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.