Structure-function relationship of adenosylhomocysteinase as a target for drug design

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To better understand the reaction mechanism of adenosylhomocysteinase and to get the probe to the molecular design of drugs targeting this enzyme, we carried out various experiments and obtained several results as follows.1. Analyses of the catalytic residues and the reaction mechanism.By x-ray crystallographic analysis advancing separately, the tertiary structure of an adenosine complex of mutated enzyme K185N-NAD^+ was solved. Conducting site-directed mutagenesis based on the structure, we succeeded in clarifying the detailed reaction mechanism of the enzyme catalysis.2. Relationship between the suicide-like reaction and the nucleosidase activity.We analyzed the stoichiometry of adenosine degradation caused by the reaction with the NADH-type enzyme. It was revealed that the product adenine was not released from the enzyme protein through the reaction, and thus it was presumed that the turnover of the enzyme had not been occurred.3. Studies on adenosylhomocysteinase-like protein.We realized that the studies on AHCYL which had been reported as adenosylhomocysteinase-like protein was useful to execute this project, since the protein is a natural mutant of the enzyme. Although it is reported that the protein had no catalytic activity of adenosylhomocysteinase, more detailed study is needed from the enzymological point of view. As a result of precise comparison of the primary structures, it became clear that the four catalytic residues and the main residues related to the coenzyme binding, all of which we had solved, were conserved in the protein. As an initial step, we established an expression system in the E. coli, purified the protein, and started protein-chemical and enzymological analysis. From the several lines of experimental evidence, we proposed a unique structure that seems to reflect the function of AHCYL.