| 摘 要： | An isoform of trichosanthin(TCS),neo－trichosanthin(n－TCS), as well as its site－directed mutant－Y70A neo－trichosanthin,has been cloned and expressed as recombinant protein. Crystals of n－TCS and Y70A n－TCSwere grown by handing drop vapor diffusion technique. X－ray diffraction data werecollected to 0.20 nm and 0.205 nm separately on a Mar Research IP. The structures weredetermined by the difference Fourier method. The final R factor are 0.183 and 0.185respectively. The r.m.s. deviations of bond length are 0.0016 nm and 0.0014 nm, the r.m.s.deviations of bond angle are 2.930° and 2.732° respectively, Although there are 11 aminoacids difference between n－TCS and TCS, all those residues are not situated in theactive pocket. In addition, most of those amino acid replacements are conservativesubstitutions. In comparison with the structure of TCS, the hydrogen bonds form betweenthe main chains of those 11 residues in n－TCS and their neighboring residues at the samepositions of TCS 11 residues within the secondary structures remain unaltered. Thereforethe overall structure of n－TCS is very similar to TCS. The crystal of Y70A n－TCS wasallowed to soak in buffer containing 5'－adenosine monophosphate (5'AMP) (10g/l) prior todata collection. No electron density corresponding to adenine can be detected around theactive pocket. Furthermore, no adenine can be detected after incubation of Y70A n－TCSwith 5'AMP. Those results suggest that Tyr70 is crucial to n－TCS and TCS for substraterecognition, binding and perhaps hydrolysis of N－glycoside bond.
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