Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190

The Journal of Biological Chemistry
Yoshimi TakataFusao Takusagawa

Abstract

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild...Continue Reading

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