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    Comparative Kinetics of Cofactor Association and Dissociation for the Human and Trypanosomal S-Adenosylhomocysteine Hydrolases. 3. Role of Lysyl and Tyrosyl Residues of the C-Terminal Extension

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    Issue Date
    2010-09-28
    Author
    Cai, Sumin
    Fang, Jianwen
    Li, Qing-Shan
    Borchardt, Ronald T.
    Kuczera, Krzysztof
    Middaugh, C. Russell
    Schowen, Richard L.
    Publisher
    ACS
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
    Rights
    Copyright © 2010 American Chemical Society
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    Abstract
    Based on 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 non-identical 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 et al., 1994 J Biol Chem, 269, 31472–8). 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 about 800 nM while TcY435A increases the cofactor equilibrium dissociation constant from approximately 100 nM (Tc-SAHH) to about 1 mM. Both changes result from larger increases in off-rate combined with smaller decreases in 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.
    URI
    http://hdl.handle.net/1808/23682
    DOI
    https://doi.org/10.1021/bi1007595
    Collections
    • Molecular Biosciences Scholarly Works [606]
    Citation
    Cai, S., Fang, J., Li, Q.-S., Borchardt, R. T., Kuczera, K., Middaugh, C. R., & Schowen, R. L. (2010). Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 3. The role of lysyl and tyrosyl residues of the C-terminal extension. Biochemistry, 49(38), 8434–8441. http://doi.org/10.1021/bi1007595

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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