Structure and Mechanism of Copper- and Nickel-Substituted Analogues of Metallo-β-lactamase L1

Authors

Zhenxin Hu, Miami University - Oxford
Lauren J. Spadafora, Miami University - Oxford
Christine E. Hajdin, Miami University - Oxford
Brian Bennett, Marquette UniversityFollow
Michael W. Crowder, Miami University - Oxford

Document Type

Article

Language

eng

Format of Original

9 p.

Publication Date

4-2009

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

doi: 10.1021/bi802295z

Abstract

In an effort to further probe metal binding to metallo-β-lactamase L1 (mβl L1), Cu- (Cu-L1) and Ni-substituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equiv of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin−spin coupling. Cu-L1 exhibited higher k_cat (96 s⁻¹) and K_m (224 μM) values, as compared to the values of dinuclear Zn(II)-containing L1, when nitrocefin was used as substrate. The Ni-L1 bound 1 equiv of Ni and 0.3 equiv of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by ¹H NMR spectra, which showed relatively sharp proton resonances. Stopped-flow kinetic studies showed that ZnNi-L1 stabilized significant amounts of the nitrocefin-derived intermediate and that the decay of intermediate is rate-limiting. ¹H NMR spectra demonstrate that Ni(II) binds in the Zn₂ site and that the ring-opened product coordinates Ni(II). Both Cu-L1 and ZnNi-L1 hydrolyze cephalosporins and carbapenems, but not penicillins, suggesting that the Zn₂ site modulates substrate preference in mβl L1. These studies demonstrate that the Zn₂ site in L1 is very flexible and can accommodate a number of different transition metal ions; this flexibility could possibly offer an organism that produces L1 an evolutionary advantage when challenged with β-lactam-containing antibiotics.

Comments

Accepted version. Biochemistry, Vol. 48, No. 13 (April 2009): 2981-2989. DOI. © 2009 American Chemical Society Publications. Used with permission.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

Recommended Citation

Hu, Zhenxin; Spadafora, Lauren J.; Hajdin, Christine E.; Bennett, Brian; and Crowder, Michael W., "Structure and Mechanism of Copper- and Nickel-Substituted Analogues of Metallo-β-lactamase L1" (2009). Physics Faculty Research and Publications. 10.
https://epublications.marquette.edu/physics_fac/10