Catalytic mechanism of S-adenosylhomocysteine hydrolase: Roles of His 54, Asp130, Glu155, Lys185, and Aspl89
| Autor: | Hirofumi Ogawa, Junichi Komoto, Tomoharu Gomi, Fusao Takusagawa, Taro Yamada, Yoshimi Takata, Motoji Fujioka |
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| Rok vydání: | 2005 |
| Předmět: |
Models
Molecular Stereochemistry Crystallography X-Ray Biochemistry Hydrolase Animals Amino Acids Binding site Histidine Bond cleavage chemistry.chemical_classification Binding Sites Molecular Structure biology Chemistry Adenosylhomocysteinase Active site Cell Biology NAD Protein Structure Tertiary Rats Amino acid Protein Subunits Enzyme Liver Mutagenesis Site-Directed biology.protein |
| Zdroj: | The International Journal of Biochemistry & Cell Biology. 37:2417-2435 |
| ISSN: | 1357-2725 |
| DOI: | 10.1016/j.biocel.2005.06.009 |
| Popis: | S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 A resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S(D) bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S(D) bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-S(D) bond of AdoHcy. |
| Databáze: | OpenAIRE |
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