Kerri D Slavens

Texas Woman's University, Denton, Texas, United States

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Publications (3)5.6 Total impact

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    ABSTRACT: The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein-protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.
    The Protein Journal 07/2014; 33(5). DOI:10.1007/s10930-014-9573-y · 1.04 Impact Factor
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    ABSTRACT: Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis-Menten constant (K(m)) was determined for all three substrates (glycine, GAB and ATP): glycine K(m) increased by 30-115-fold, GAB K(m) decreased by 8-17-fold, and the ATP K(m) was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.
    Biochemical and Biophysical Research Communications 08/2011; 411(3):536-42. DOI:10.1016/j.bbrc.2011.06.166 · 2.28 Impact Factor
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    ABSTRACT: It was hypothesized that residues Val44 and Val45 serve as important residues for human glutathione synthetase (hGS) function and stability given their location at the dimer interface of this enzyme. Computational studies suggest that mutation at Val45 has more impact on the structure and stability of hGS than does mutation at Val44. Experimentally, enzymes with mutations at the 44 and or 45 positions of hGS were prepared, purified and assayed for initial activity. Val45 position mutations (either to alanine or tryptophan) have a greater impact on enzyme activity than do mutations at Val44. Differential scanning calorimetry experiments reveal a loss of stability in all mutant enzymes, with V45 mutations being less stable than the corresponding Val44 mutations. The γ-GluABA substrate affinity remains unaltered in V44A and V45A mutant enzymes, but increases when tryptophan is introduced at either of these positions. Hill coefficients trend towards less negative cooperativity with the exception of V45W mutant hGS. These results imply that residues V44 and V45 are located along the allosteric pathway of this negatively cooperative dimeric enzyme, that their mutation impacts the allosteric pathway more than it does the active site of hGS, and that these residues (and by extension the dimer interface in which they are located) are integral to the stability of human glutathione synthetase.
    Biochemical and Biophysical Research Communications 06/2011; 410(3):597-601. DOI:10.1016/j.bbrc.2011.06.034 · 2.28 Impact Factor