Motion of the zinc ions in catalysis by a dizinc metallo-beta-lactamase

Abstract

We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-beta-lactamase (MbetaL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MbetaL move away from each other, by approximately 0.3 A after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 A. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 A, these data indicate a scissoring motion of the active site that accompanies the ring-opening step. The average Zn(II) coordination number of 4.5 in the resting enzyme appears to be maintained throughout the reaction with nitrocefin. This is the first direct structural information available on early stage dizinc metallo-beta-lactamase catalysis.

Figure 1

Summary of the time-dependent structure of resting ZnZn-L1 (left), freeze quenched after 10 ms of reaction with nitrocefin (center) and the resulting product complex (right) derived from EXAFS. The structure of nitrocefin, shown above the resting structure, is truncated in the 10 ms and product structures. The Zn-Zn separations are given below each structure.

Figure 2

Comparison of the Fourier transformed EXAFS of ZnZn-L1, freeze quenched after 10 ms of reaction with nitrocefin (bold line), with resting ZnZn-L1 (top) and the ZnZn-L1 product complex with nitrocefin (center), and the best fit to the data (bottom). The data for ZnZnL1 and the ZnZnL1-nitrocefin product complex are reproduced, for comparison, from Costello, et al.