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Kinetic and Structural Characterization of Manganese(II)-Loaded Methionyl Aminopeptidases
Biochemistry
  • Ventris M. D'Souza, Utah State University
  • Sabina I. Swierczek, Utah State University
  • Nathaniel J. Cosper, University of Georgia
  • Lu Meng, Utah State University
  • Shane Ruebush, Utah State University
  • Alicja J. Copik, Utah State University
  • Robert A. Scott, University of Georgia
  • Richard C. Holz, Marquette University
Document Type
Article
Publication Date
10-4-2002
Disciplines
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 (Kd) for the first metal binding site were found to be 6 ± 0.5 and 1 ± 0.5 μM for EcMetAP-I and PfMetAP-II, respectively. These Kd values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 ± 0.2 and 1.4 ± 0.2 μM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 °C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and Km 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 °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 °C are ΔG⧧ = 50.1 kJ/mol, ΔH⧧ = 23.2 kJ/mol, and ΔS⧧ = −90.2 J·mol-1·K-1.
Comments

Biochemistry, Vol. 41, No. 43 (October 4, 2002): 13096-13105. DOI.

Richard Holz was affiliated with the Utah State University at the time of publication.

Citation Information
Ventris M. D'Souza, Sabina I. Swierczek, Nathaniel J. Cosper, Lu Meng, et al.. "Kinetic and Structural Characterization of Manganese(II)-Loaded Methionyl Aminopeptidases" Biochemistry (2002) ISSN: 0006-2960
Available at: http://works.bepress.com/richard_holz/48/