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10 p.

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American Chemical Society

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doi: 10.1021/bi060893t


In an effort to understand the reaction mechanism of a B2 metallo-β-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0−8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s-1 and a slower rate of fluorescence regain of 98 s-1. Stopped-flow UV−vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s-1. These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C−N bond cleavage. Rapid freeze quench EPR studies on CoII-substituted ImiS demonstrated the appearance of a rhombic signal after 10 ms that is assigned to a reaction intermediate that has a five-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18−19 ms. When these results are taken together, they allow for a reaction mechanism to be offered for the B2 metallo-β-lactamases and demonstrate that the mono- and dinuclear ZnII-containing enzymes share a common rate-limiting step, which is C−N bond cleavage.


Accepted version. Biochemistry, Vol. 45, No. 35 (September 2006): 10729-10738. DOI. © 2006 American Chemical Society Publications. Used with permission.

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

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