First-Sphere and Second-Sphere Electrostatic Effects in the Active Site of a Class Mu Glutathione Transferase
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- 作者：Gaoyi Xiao ; Suxing Liu ; Xinhua Ji ; William W. Johnson ; Jihong Chen ; James F. Parsons ; Walter J. Stevens ; Gary L. Gilliland ; and Richard N. Armstrong
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：April 16, 1996
- 年：1996
- 卷：35
- 期：15
- 页码：4753 - 4765
- 全文大小：654K
- 年卷期：v.35,no.15(April 16, 1996)
- ISSN：1520-4995
文摘
The activation of the thiol of glutathione (GSH) bound in theactive site of the class muglutathione transferase M1-1 from rat involves a hydrogen-bondingnetwork that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur ofGSH and second-sphere interactionsinvolving a hydrogen bond between the main-chain amide N-H of L12 andthe hydroxyl group of Y6 andan on-face hydrogen bond between the hydroxyl group of T13 and the
pi.gif" BORDER=0 >-electron cloud of Y6 (i.e., T13-OH- - -pi.gif" BORDER=0 >-Y6-OH- - --SG). The functions ofthese hydrogen bonds have been examined with a combinationof site-specific mutagenesis and X-ray crystallography. Thehydroxyl group of Y6 has a normal pKaofabout 10 even though it is shielded from solvent and is in a largelyhydrophobic environment. The apparentpKa of GSH in the binary Y6F·GSH complexis increased by 1.6 log units, and the reactivity of theenzyme-bound nucleophile is reduced. The catalytic properties ofthe Y6L mutant are identical to thoseof Y6F, suggesting that the weakly polar on-edge interaction betweenthe aromatic ring and sulfur has noinfluence on catalysis. The refined three-dimensional structure ofthe Y6F mutant in complex with GSHshows no major structural perturbation of the protein other than achange in the coordination environmentof the sulfur. Removal of the second-sphere influence of theon-face hydrogen bond between the hydroxylgroup of T13 as in the T13V and T13A mutants elevates thepKa of enzyme-bound GSH by about 0.7pKaunits. Crystal structures of these mutants show that structuralchanges in the active site are minor andsuggest that the changes in pKa of E·GSHare due to the presence or absence of the on-face hydrogenbond. The T13S mutant has a completely different side-chainhydrogen-bonding geometry than T13 inthe native enzyme and catalytic properties similar to the T13A and T13Vmutants consistent with theabsence of an on-face hydrogen bond. The 
-methyl group of T13is essential in enforcing the on-facehydrogen bond geometry and preventing the hydroxyl group from formingmore favorable conventionalhydrogen bonds.
pi.gif" BORDER=0 >-electron cloud of Y6 (i.e., T13-OH- - -pi.gif" BORDER=0 >-Y6-OH- - --SG). The functions ofthese hydrogen bonds have been examined with a combinationof site-specific mutagenesis and X-ray crystallography. Thehydroxyl group of Y6 has a normal pKaofabout 10 even though it is shielded from solvent and is in a largelyhydrophobic environment. The apparentpKa of GSH in the binary Y6F·GSH complexis increased by 1.6 log units, and the reactivity of theenzyme-bound nucleophile is reduced. The catalytic properties ofthe Y6L mutant are identical to thoseof Y6F, suggesting that the weakly polar on-edge interaction betweenthe aromatic ring and sulfur has noinfluence on catalysis. The refined three-dimensional structure ofthe Y6F mutant in complex with GSHshows no major structural perturbation of the protein other than achange in the coordination environmentof the sulfur. Removal of the second-sphere influence of theon-face hydrogen bond between the hydroxylgroup of T13 as in the T13V and T13A mutants elevates thepKa of enzyme-bound GSH by about 0.7pKaunits. Crystal structures of these mutants show that structuralchanges in the active site are minor andsuggest that the changes in pKa of E·GSHare due to the presence or absence of the on-face hydrogenbond. The T13S mutant has a completely different side-chainhydrogen-bonding geometry than T13 inthe native enzyme and catalytic properties similar to the T13A and T13Vmutants consistent with theabsence of an on-face hydrogen bond. The -methyl group of T13is essential in enforcing the on-facehydrogen bond geometry and preventing the hydroxyl group from formingmore favorable conventionalhydrogen bonds.