| dc.description.abstract | In both mammals and parasites, S-adenosyl-L-homocysteine hydrolase (SAHH) plays a crucial role in regulating S-adenosylmethionine dependent transmethylations by catalyzing the reversible conversion of adenosyl-L-homocysteine (AdoHcy) to adenosine and homocysteine. Inhibitors of parasite SAHHs (e.g., those of Leishmania, Plasmodium, Trypanosoma) are potential anti-parasitic agents which may present selective inhibition if their inhibitory activity is weaker for Homo sapiens SAHH (Hs-SAHH). This study was aimed to address the differences in structure and kinetic properties between Hs-SAHH and Tc-SAHH, the SAHH of Trypanosoma cruzi, the organism which causes Chagas disease in humans. The available X-ray structures of Hs-SAHH and Tc-SAHH show no significant difference between the active sites of the two enzymes, providing no definite suggestions for designing selective anti-parasitic inhibitors. Therefore, we have designed a series of biochemical, biophysical and computational studies focusing on enzyme interactions with the nicotinamide cofactors. These studies showed that the equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Hs-SAHH and Tc-SAHH are qualitatively similar but quantitatively distinct. Briefly, association of NAD+ to both enzymes is complex process, composed of two parts: a fast-binding phase (dead time) and a slow-binding phase. The fast-binding phase comes from one class of the homotetrameric apo-enzyme active sites, which binds cofactor weakly and generates full activity very rapidly (in less than a minute). The slow-binding phase comes from the other class of active sites which binds cofactor more strongly but generates activity only slowly (over 30 min). These two classes of active sites appear to be numerically equal. The kinetics of slow binding of NAD+ to two enzymes show concentration dependence, and the cofactor binds to Hs-SAHH almost 10 times faster than to Tc-SAHH. The final binding affinity of cofactor NAD+ to Tc-SAHH persists at micromolar level while Hs-SAHH decreases the binding affinity from micromolar to nanomolar over a period of time as its equilibrium affinity. In contrast to the complex kinetics of association, both enzymes undergo dissociation of NAD+ from all four sites in a single first-order reaction. The dissociation of NAD+ from two enzymes shows complex temperature dependence and NAD+ leaves from Tc-SAHH much faster than from Hs-SAHH. Compared to the traditional selective inhibitor design site (the substrate-binding domain of SAHH), the identified differential features between Hs-SAHH and Tc-SAHH suggest the use of a novel design site, the cofactor (NAD+/NADH)-binding domain. In a detailed analysis, two structural elements, the helix 18 at the C-terminal and the β-sheet A of the Rossmann motif in the cofactor-binding domain, are identified as responsible for the distinction in properties between the two enzymes. The site-directed mutagenesis approach creates two kinds of mutants: one is the "humanized" Tc-SAHH (helix 18 or β-sheet A of Tc-SAHH replaced by that of human enzyme) and the other one is the "parasitized" Hs-SAHH (helix 18 or β-sheet A of Hs-SAHH replaced by that of parasite enzyme). As expected, the results for the two mutants were intermediate between the two wild types: the "humanized" Tc-SAHH exhibits similar kinetics and thermodynamics to wild type Hs-SAHH while the "parasitized" Hs-SAHH has properties close to wild type parasite enzymes. Moreover, in the alanine scanning computational study, two conserved residues at the C-terminus, a Lys and a Tyr, are found to be involved in differential properties of the two enzymes. All these data support the view that the cofactor-binding domain is a good target for designing selective inhibitors against parasite enzymes. In addition, this work found a selective inhibitor binding to the traditional design site - the substrate binding site, and studied its inactivation mechanism and kinetic features. Ribavirin, an analogue of adenosine, exhibits preferential time-dependent inactivation on Tc-SAHH over Hs-SAHH and provides a structural lead to design more selective inhibitors. Overall, studies on differential cofactor association and dissociation properties between Hs-SAHH and Tc-SAHH will help us understand better on these two enzymes and lead to design potential selective inhibitors targeting at cofactor-binding sites for treatment of Chagas disease. | |
