8 予期しない基質特異性を有する触媒抗体の活性部位解析(口頭発表の部)

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According to Schultz's procedure, immunization of the KLH-phosphonate 1 adduct 2 afforded 34 IgG's. After purification by standard protocols, of the 34 antibodies isolated, 4A1, 5H2, and 1G2 were found to have catalytic activities for carbonate 3a with values of kcat/kuncat of 3×10^3, 450, and 440 at 37 C, pH 8.5, respectively. In the examination of substrate specificity for these abzymes in the vicinity of scissile bond, however, one of antibodies, 4A1 showed unpredicted rate acceleration for (S)-13 having 2,2-dimethyl-1,3-dioxolane group with value of kcat/kuncat of 6.4×10^4. Similar propensity was also observed in the case of the R and S isomers of 13-15 subjected to significant changes in structure around the scissile bond. When these values are compared with 3.7×10^3 for carbonate 6 most similar to the induced hapten structure, it is indicated that an accepted propensity, which demand fairly strict substrate homology to the hapten, is not always preserved in this case. In order to explain the unprecedented substrate specificity for 4A1 antibody, several kinetic parameters (km, kcat, kcat/km) and dissociation constants (kd) for various substrates and transition state analogs were determined. These analyses were strongly suggested that another binding element should exist in the vicinity of 4A1 combining site. This assumption was finaly demonstrated by product inhibition of substrate 22 containing a-acetylamino phenetyl group. Thus, we have demonstrated that 4A1 antibody has an unexpected binding element(s) in close proximity to the active site. It is probable that large catalytic efficiency for the modified substrates is due to cooperative influences between the active site and the subsite,
天然有機化合物討論会の論文
1992-09-10

8 予期しない基質特異性を有する触媒抗体の活性部位解析(口頭発表の部)

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