title: Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli.
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 approximately 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 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.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA.
authors.forename: Sanghamitra
authors.initials: S
authors.lastname: Mitra
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Job
authors.forename: Lu
authors.initials: L
authors.lastname: Meng
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Richard C
authors.initials: RC
authors.lastname: Holz
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine; D001224:Aspartic Acid; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid; D005998:Glycine; D007700:Kinetics; D008715:Methionine; D063208:Methionyl Aminopeptidases; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D008715:Methionine; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; C022750:X-Pro aminopeptidase; D000409:Alanine; D005998:Glycine