title: Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases.
abstract: Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
authors.forename: Ventris M
authors.initials: VM
authors.lastname: D'souza
authors.forename: Sabina I
authors.initials: SI
authors.lastname: Swierczek
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Cosper
authors.forename: Lu
authors.initials: L
authors.lastname: Meng
authors.forename: Shane
authors.initials: S
authors.lastname: Ruebush
authors.forename: Alicja J
authors.initials: AJ
authors.lastname: Copik
authors.forename: Robert A
authors.initials: RA
authors.lastname: Scott
authors.forename: Richard C
authors.initials: RC
authors.lastname: Holz
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D063208:Methionyl Aminopeptidases; D009842:Oligopeptides / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013696:Temperature; D014965:X-Rays
chemical_list: C467041:N-acetyl-methionyl-alanyl-serine; D009842:Oligopeptides; D008345:Manganese; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases