title: A single mutation at lysine 426 of human placental S-adenosylhomocysteine hydrolase inactivates the enzyme.
abstract: S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy), as well as the reverse reaction, through a mechanism involving an NAD(+)-dependent oxidation of the 3'-hydroxyl group of AdoHcy (3'-oxidative activity), followed by elimination of Hcy to form 3'-keto-4',5'-didehydro-5'-deoxy-Ado. The addition of water at the 5'-position (5'-hydrolytic activity) of this tightly bound intermediate, followed by an NADH-dependent reduction, results in the formation of Ado. Based on a computer graphics model of the active site of this enzyme, it was hypothesized that amino acid residues at the carboxyl-terminal end of the protein reside in the active site of the enzyme and could play a role in catalyzing the 5'-hydrolytic reaction (Yeh, J. C., Borchardt, R. T., and Vedani, A. (1991) J. Comput. Aided Mol. Des. 5, 213-234). Using site-directed mutagenesis, we show here that lysine 426 is essential for the catalytic activity of the enzyme and that it appears to play a crucial role in the 5'-hydrolytic activity and/or stability of the quaternary structure of the human placental enzyme. Mutation of Lys-426 to arginine (K426R) produces a stable tetrameric enzyme that lacks overall catalytic activity and that was isolated predominantly as its NADH form containing tightly bound 3'-keto-Ado, suggesting that the K426R mutant has oxidative activity, but lacks 5'-hydrolytic activity, preventing it from completing the entire catalytic cycle. Mutations of Lys-426 to glutamic acid (K426E) and alanine (K426A) produce enzymes that exist primarily as monomers, do not bind NAD+ or NADH, and lack catalytic activity. The results of the Lys-426 mutations suggest that this lysine residue is crucial for the 5'-hydrolytic activity of the enzyme and/or stabilizing the quaternary structure of the enzyme.
authors.affiliation: Department of Biochemistry, University of Kansas, Lawrence 66045.
authors.forename: D B
authors.initials: DB
authors.lastname: Ault-Riché
authors.forename: C S
authors.initials: CS
authors.lastname: Yuan
authors.forename: R T
authors.initials: RT
authors.lastname: Borchardt
mesh_terms: D043383:Adenosylhomocysteinase; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D006867:Hydrolases / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D011485:Protein Binding; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D046911:Macromolecular Substances; D001120:Arginine; D012435:S-Adenosylhomocysteine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D008239:Lysine