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Kinetic And Spectroscopic Analysis Of The Catalytic Role Of H79 In The Methionine Aminopeptidase From Escherichia Coli, Sarah J. Watterson, Sanghamitra Mitra, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Kinetic And Spectroscopic Analysis Of The Catalytic Role Of H79 In The Methionine Aminopeptidase From Escherichia Coli, Sarah J. Watterson, Sanghamitra Mitra, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Physics Faculty Research and Publications
To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in kcat. Interestingly, the Km value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, kcat/Km values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded …
Metal Content Of Metallo-Β-Lactamase L1 Is Determined By The Bioavailability Of Metal Ions, Zhenxin Hu, Thusitha S. Gunasekera, Lauren J. Spadafora, Brian Bennett, Michael W. Crowder
Metal Content Of Metallo-Β-Lactamase L1 Is Determined By The Bioavailability Of Metal Ions, Zhenxin Hu, Thusitha S. Gunasekera, Lauren J. Spadafora, Brian Bennett, Michael W. Crowder
Physics Faculty Research and Publications
In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized …