The GI mutant, GIK253RA198C, was obtained by in vitro site-directed mutagenesis using the double primer method and then expressed in E.coli K38. The characteristic analysis of the enzymes showed that thermostability of GIK253RA198C was significantly lower as compared with wild-type GI in 80℃. Moreover, its optimum reaction temperature decreased from 75℃ to 70℃. The results are explained by the kinetic parameters. Previous experiment indicated that the same mutation of K253R produced different effects on the thermostablity of SM33 GI and A.missouriensis GI. It is clarified here, based on structure and mechanism, that the result was due to the minor difference between Lys253's positions in the two GI structures.