The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cellfree extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of～48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD1 in the incubation medium. The dissociation constant (Kd) for NAD1 with the L. donovani enzyme was estimated to be 2.1±0.2μM. The Km values for the natural substrates of theenzyme, AdoHcy, Ado, and Hcy, were determined tobe 21±3, 8±2, and 82±5μM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design speci®c inhibitors of this parasitic enzyme as potential antiparasitic agents.

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 Kd of～10-11M. The association rate constant was determined to be 7×10-6 M-1 s-1. The releasing of copper ions from the enzyme was found to be biphasic with a k(1) of 2.8×10-3 s-1 and k(2) of 1.7×10-5 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 K1=8.0×10-5M and result in an increased affinity (Kd=～10-17M) of binding of copper ions to the enzyme.

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