Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli. FEBS J 2008 Dec;275(24):6248-59
Date
11/21/2008Pubmed ID
19019076Pubmed Central ID
PMC2699115DOI
10.1111/j.1742-4658.2008.06749.xScopus ID
2-s2.0-56849108463 (requires institutional sign-in at Scopus site) 8 CitationsAbstract
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.
Author List
Mitra S, Job KM, Meng L, Bennett B, Holz RCAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
AlanineAmino Acid Sequence
Amino Acid Substitution
Aminopeptidases
Asparagine
Aspartic Acid
Catalysis
Catalytic Domain
Escherichia coli
Escherichia coli Proteins
Glutamic Acid
Glycine
Kinetics
Methionine
Methionyl Aminopeptidases
Models, Molecular
Molecular Conformation
Molecular Sequence Data
Recombinant Proteins
Sequence Alignment
Substrate Specificity
