title: Binding of Cu2+ to S-adenosyl-L-homocysteine hydrolase.
abstract: S-Adenosylhomocysteine (AdoHcy) hydrolase regulates biomethylation and homocysteine metabolism. It has been proposed to be a copper binding protein playing an important role in copper transport and distribution. In the present work, the kinetics of binding and releasing of copper ions was studied using fluorescence method. The dissociation constant for copper ions with AdoHcy hydrolase was determined by fluorescence quenching titration and activity titration methods using ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and glycine as competitive chelators. The experimental results showed that copper ions bind to AdoHcy hydrolase with a K(d) of approximately 10(-11) M. The association rate constant was determined to be 7 x 10(6) M(-1)s(-1). The releasing of copper ions from the enzyme was found to be biphasic with a k(1) of 2.8 x 10(-3) s(-1) and k(2) of 1.7x10(-5) s(-1). It is suggested that copper ions do not bind to the substrate binding sites because the addition of adenine substrate did not compete with the binding of copper to AdoHcy hydrolase. Interestingly, it was observed that EDTA could bind to AdoHcy hydrolase with a dissociation constant of K(1) = 8.0 x 10(-5) M and result in an increased affinity (K(d) = approximately 10(-17) M) of binding of copper ions to the enzyme.
authors.affiliation: Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083, PR China.
authors.forename: Yanjie
authors.initials: Y
authors.lastname: Li
authors.forename: Jiejin
authors.initials: J
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Xiaoda
authors.initials: X
authors.lastname: Yang
authors.forename: Kui
authors.initials: K
authors.lastname: Wang
mesh_terms: D043383:Adenosylhomocysteinase / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding
chemical_list: D003300:Copper; D043383:Adenosylhomocysteinase