Qing-Shan Li

University of Kansas, Lawrence, KS, United States

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Publications (6)14.46 Total impact

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    ABSTRACT: On the basis of the available X-ray structures of S-adenosylhomocysteine hydrolases (SAHHs), free energy simulations employing the MM-GBSA approach were applied to predict residues important to the differential cofactor binding properties of human and trypanosomal SAHHs (Hs-SAHH and Tc-SAHH), within 5 Å of the cofactor NAD(+)/NADH binding site. Among the 38 residues in this region, only four are different between the two enzymes. Surprisingly, the four nonidentical residues make no major contribution to differential cofactor binding between Hs-SAHH and Tc-SAHH. On the other hand, four pairs of identical residues are shown by free energy simulations to differentiate cofactor binding between Hs-SAHH and Tc-SAHH. Experimental mutagenesis was performed to test these predictions for a lysine residue and a tyrosine residue of the C-terminal extension that penetrates a partner subunit to form part of the cofactor binding site. The K431A mutant of Tc-SAHH (TcK431A) loses its cofactor binding affinity but retains the wild type's tetrameric structure, while the corresponding mutant of Hs-SAHH (HsK426A) loses both cofactor affinity and tetrameric structure [Ault-Riche, D. B., et al. (1994) J. Biol. Chem. 269, 31472-31478]. The tyrosine mutants HsY430A and TcY435A alter the NAD(+) association and dissociation kinetics, with HsY430A increasing the cofactor equilibrium dissociation constant from approximately 10 nM (Hs-SAHH) to ∼800 nM and TcY435A increasing the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to ∼1 mM. Both changes result from larger increases in the off rate combined with smaller decreases in the on rate. These investigations demonstrate that computational free energy decomposition may be used to guide experimental studies by suggesting sensitive sites for mutagenesis. Our finding that identical residues in two orthologous proteins may give significantly different binding free energy contributions strongly suggests that comparative studies of homologous proteins should investigate not only different residues but also identical residues in these proteins.
    Biochemistry 09/2010; 49(38):8434-41. · 3.38 Impact Factor
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    ABSTRACT: Trypanosomal S-adenoyl-L-homocysteine hydrolase (Tc-SAHH), considered as a target for treatment of Chagas disease, has the same catalytic mechanism as human SAHH (Hs-SAHH) and both enzymes have very similar x-ray structures. Efforts toward the design of selective inhibitors against Tc-SAHH targeting the substrate binding site have not to date shown any significant promise. Systematic kinetic and thermodynamic studies on association and dissociation of cofactor NAD/H for Tc-SAHH and Hs-SAHH provide a rationale for the design of anti-parasitic drugs directed toward cofactor-binding sites. Analogues of NAD and their reduced forms show significant selective inactivation of Tc-SAHH, confirming that this design approach is rational.
    Nucleosides Nucleotides &amp Nucleic Acids 05/2009; 28(5):485-503. · 0.71 Impact Factor
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    ABSTRACT: S-Adenosylhomocysteine (AdoHcy) hydrolases (SAHHs) from human sources (Hs-SAHHs) bind the cofactor NAD(+) more tightly than several parasitic SAHHs by around 1000-fold. This property suggests the cofactor binding site of this essential enzyme as a potential anti-parasitic drug target, e.g., against SAHH from Trypansoma cruzi (Tc-SAHH). The on-rate and off-rate constants and the equilibrium dissociation constants were determined for NAD(+)/NADH analogues and suggested that NADH analogues were the most promising for selective inhibition of Tc-SAHH. None significantly inhibited Hs-SAHH while S-NADH and H-NADH (see Figure 1) reduced the catalytic activity of Tc-SAHH to < 10% in six minutes of exposure.
    Nucleosides Nucleotides &amp Nucleic Acids 05/2009; 28(5):473-84. · 0.71 Impact Factor
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    ABSTRACT: The S-adenosyl- l-homocysteine (AdoHcy) hydrolases (SAHH) from Homo sapiens (Hs-SAHH) and from the parasite Trypanosoma cruzi (Tc-SAHH) are very similar in structure and catalytic properties but differ in the kinetics and thermodynamics of association and dissociation of the cofactor NAD (+). The binding of NAD (+) and NADH in SAHH appears structurally to be mediated by helix 18, formed by seven residues near the C-terminus of the adjacent subunit. Helix-propensity estimates indicate decreasing stability of helix 18 in the order Hs-SAHH > Tc-SAHH > Ld-SAHH (from Leishmania donovani) > Pf-SAHH (from Plasmodium falciparum), which would be consistent with the previous observations. Here we report the properties of Hs-18Pf-SAHH, the human enzyme with plasmodial helix 18, and Tc-18Hs-SAHH, the trypanosomal enzyme with human helix 18. Hs-18Tc-SAHH, the human enzyme with trypanosomal helix 18, was also prepared but differed insignificantly from Hs-SAHH. Association of NAD (+) with Hs-SAHH, Hs-18Pf-SAHH, Tc-18Hs-SAHH, and Tc-SAHH exhibited biphasic kinetics for all enzymes. A thermal maximum in rate, attributed to the onset of local structural alterations in or near the binding site, occurred at 35, 33, 30, and 15 degrees C, respectively. This order is consistent with some reversible changes within helix 18 but does require influence of other properties of the "host enzyme". Dissociation of NAD (+) from the same series of enzymes also exhibited biphasic kinetics with a transition to faster rates (a larger entropy of activation more than compensates for a larger enthalpy of activation) at temperatures of 41, 38, 36, and 29 degrees C, respectively. This order is also consistent with changes in helix 18 but again requiring influence of other properties of the "host enzyme". Global unfolding of all fully reconstituted holoenzymes occurred around 63 degrees C, confirming that the kinetic transition temperatures did not arise from a major disruption of the protein structure.
    Biochemistry 04/2008; 47(17):4983-91. · 3.38 Impact Factor
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    ABSTRACT: Ribavirin (1,2,4-triazole-3-carboxamide riboside) is a well-known antiviral drug. Ribavirin has also been reported to inhibit human S-adenosyl-L-homocysteine hydrolase (Hs-SAHH), which catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and homocysteine. We now report that ribavirin, which is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and Trypanosoma cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD(+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K(I) values (266 and 194 microM), but the slow inactivation step is 5-fold faster with Tc-SAHH. Ribavirin may provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs.
    Bioorganic & Medicinal Chemistry 01/2008; 15(23):7281-7. · 2.90 Impact Factor
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    ABSTRACT: The S-adenosyl-l-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion of AdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+ oxidizes the 3-hydroxyl group of the substrate at the beginning of the cycle, activating the 4-CH bond for elimination of homocysteine, followed by Michael addition of water to the resulting intermediate and a final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic properties of the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH) and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymes bind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear to divide into two numerically equal classes of active sites. One class of sites binds cofactor weakly and generates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly but generates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughly micromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rate constant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding of NAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however, binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for binding to Tc-SAHH levels out at 0.006 s-1. 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 equilibrium affinities of both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constants and the slow-binding association rate constants for NAD+ show a complex temperature dependence with both enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 degrees C, and the cofactor binds more rapidly to Hs-SAHH than to Tc-SAHH above approximately 16 degrees C. These features present an opening for selective inhibition of Tc-SAHH over Hs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind to Hs-SAHH with approximately 40 nM affinities but much more weakly to Tc-SAHH (0.6-15 microM). Nevertheless, both analogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, while the degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equal to the rate of dissociation of the cofactor and thus 80-fold faster at 37 degrees C for Tc-SAHH.
    Biochemistry 05/2007; 46(19):5798-809. · 3.38 Impact Factor