Detailed structure analysis of active site of -lactamase TOHO-1

-lactamase TOHO-1活性部位の詳細構造解析

栗原 和男

Kurihara, Kazuo

To help resolve questions regarding the catalytic activity of -lactamase, the crystal structure of an unliganded form of the -lactamase Toho-1 with double mutation R274N/R276N (Toho-1/NN) has been determined by the use of high-resolution neutron and X-ray diffraction data. The double mutation was introduced to improve the diffraction quality of Toho-1 because the wild type Toho-1 tends to form merohedrally twinned crystals. A large single crystal of Toho-1/NN with a dimension of 2.6 2.5 1.3 mm was used to collect both 100 K neutron diffraction data up to 1.5 resolution and X-ray diffraction data up to 1.4 resolution. The structural model of Toho-1/NN was refined to an -factor of 19.7% using the program . The Fourier map showed that Glu166, a catalytic residue of Toho-1, was protonated even at pD 7 in spite of the close location to the positively charged side-chain amino group (-NH3) of Lys73. It is also found that there is the hydration water network bridging between the protonated Glu166 and the oxyanion hole comprising two main-chain nitrogen atoms of Ser70 and Ser237. The neutron structure analysis also revealed the clear configuration of the proposed catalytic water molecule bridging Glu166 and Ser70. These observations are important to understand the catalytic action of -lactamase Toho-1.