Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases.
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).
D'souza
Ventris M
VM
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Swierczek
Sabina I
SI
Cosper
Nathaniel J
NJ
Meng
Lu
L
Ruebush
Shane
S
Copik
Alicja J
AJ
Scott
Robert A
RA
Holz
Richard C
RC
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
C467041:N-acetyl-methionyl-alanyl-serine; D009842:Oligopeptides; D008345:Manganese; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases