Contribution of aromatic-aromatic interactions to the anomalous pKa of tyrosine-9 and the C-terminal dynamics of glutathione S-transferase A1-1

被引:41
作者
Ibarra, C [1 ]
Nieslanik, BS [1 ]
Atkins, WM [1 ]
机构
[1] Univ Washington, Dept Med Chem, Seattle, WA 98195 USA
关键词
D O I
10.1021/bi010672h
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Most cytosolic glutathione S-transferases (GSTs) exploit a hydrogen bond between an active site Tyr and the bound glutathione (GSH) cofactor to lower the pK(a) of the GSH and generate the nucleophilic thiolate anion. GS(-). In human (hGSTA1-1) and rat (rGSTA1-1) homologues, the active site Tyr-9 has a low pK(a), of 8.1-8.3, for which the functional significance is unknown. Crystal structures of GSTA1-1 suggest that weakly polar interactions between the electropositive ring edge of Phe-10 and the pi -cloud of Tyr-9, in the apoenzyme, could stabilize the tyrosinate anion and also modulate the pK(a) of GSH. Upon binding a product GSH conjugate, Phe-10 moves away from Tyr-9, allowing the highly dynamic C-terminus to "close" over the active site. To explore the role of Phe-10 in modulating the Tyr-9 pK(a) and in ligand binding, rGSTA1-1 mutants F10Y, F10L, and F10A were characterized. The pK(a)s of Tyr-9 in the apoenzymes were 8.2 +/- 0.2, 8.7 +/- 0.2, and 9.3 +/- 0.1, respectively, for F10Y, F10L, and F10A, compared to 8.3 +/- 0.2 for the "wild type". The experimentally determined pK(a)s qualitatively paralleled the energies required to remove a proton predicted by ab initio calculations using model compounds constrained to the coordinates of rGSTA1-1. The pK(a) of GSH in the binary complex was significantly less affected by these substitutions. In contrast, F220I and F220Y C-terminal mutations caused the pK(a) of Tyr-9 to decrease modestly. For the binary complex with S-hexyl-GSH, which induces the "closed" conformation, Tyr-9 retains a low pK(a) and the Phe-10 substitutions have significant effects. Presumably, Phe-10 plays a critical structural role in stabilizing the closed conformation. The mutations F10L and F10A also slowed the rate of GSH conjugate binding by 10-20-fold, as measured by stopped-flow fluorescence. The effects of Phe- 10 substitution were large for both steps of the biphasic binding reaction, suggesting the importance of aromatic interactions throughout the reaction coordinate. A unified view of the C-terminal dynamics of GSTA1-1 is discussed. which emphasizes the coupling between Tyr-9 ionization, active site solvation, and C-terminal dynamics.
引用
收藏
页码:10614 / 10624
页数:11
相关论文
共 45 条
[1]  
Adman ET, 2001, PROTEINS, V42, P192, DOI 10.1002/1097-0134(20010201)42:2<192::AID-PROT60>3.0.CO
[2]  
2-#
[3]   The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases [J].
Allardyce, CS ;
McDonagh, PD ;
Lian, LY ;
Wolf, CR ;
Roberts, GCK .
BIOCHEMICAL JOURNAL, 1999, 343 :525-531
[4]  
ALLARDYCE CS, 2000, CLIN CHEM ENZYMOL CO, V8, P239
[5]   Mechanistic imperatives for the evolution of glutathione transferases [J].
Armstrong, RN .
CURRENT OPINION IN CHEMICAL BIOLOGY, 1998, 2 (05) :618-623
[6]  
Atkins WM, 1997, PROTEIN SCI, V6, P873
[7]  
ATKINS WM, 1993, J BIOL CHEM, V268, P19188
[8]   SUPRAMOLECULAR SELF-ASSEMBLY OF ESCHERICHIA-COLI GLUTAMINE-SYNTHETASE - EFFECTS OF PRESSURE AND ADENYLYLATION STATE ON DODECAMER STACKING [J].
ATKINS, WM .
BIOCHEMISTRY, 1994, 33 (50) :14965-14973
[9]  
BJORNESTEDT R, 1995, J MOL BIOL, V247, P765
[10]   Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products [J].
Bruns, CM ;
Hubatsch, I ;
Ridderström, M ;
Mannervik, B ;
Tainer, JA .
JOURNAL OF MOLECULAR BIOLOGY, 1999, 288 (03) :427-439