Rational modulation of the catalytic activity of A1-1 glutathione S-transferase: Evidence for incorporation of an on-face (pi center dot center dot center dot HO-Ar) hydrogen bond at tyrosine-9
Rational modulation of the catalytic activity of A1-1 glutathione S-transferase: Evidence for incorporation of an on-face (pi center dot center dot center dot HO-Ar) hydrogen bond at tyrosine-9
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DOI：
10.1021/bi961073r
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发表时间：
1996-09-17
期刊：
影响因子：
2.9
通讯作者：
Atkins, WM
中科院分区：
文献类型：
--
作者：
Dietze, EC;Ibarra, C;Atkins, WM
The alpha-, pi-, and mu-class glutathione S-transferases utilize a hydrogen bond between a conserved tyrosine and glutathione (GSH) to stabilize the nucleophilic thiolate anion, as Tyr-OH ...(-)SG. This hydrogen bond is critical for efficient detoxication catalysis. The detailed structure of this hydrogen bond. however, is controlled by active site features which art: not conserved across class boundaries. The alpha-class GST A1-1 has a cluster of aromatic residues on one side of the ring of the catalytic tyrosine, Tyr-9, Also, a hydrophobic Met-16 side chain is packed against the edge of the ring of Tyr-9. Molecular modeling and ab initio calculations suggested that substitution of Phe-220 with tyrosine could generate an aromatic on-face hydrogen bond (pi ... HO-Ar) between the ring of Tyr-9 and the hydroxyl group of Tyr-220, and this would lower the pK(a) of enzyme-bound GSH. Therefore, Phe-220 was replaced by Tyr in the rat A1-1 isozyme. Also, Met-16 was replaced by Thr in order to investigate the effect of a hydrogen bond donor at the Tyr-9 ring edge. UV spectroscopic titration of GST GSM and steady-state kinetic analysis indicate that substitution of Tyr at Phe-220 results in a decrease of the pK(a) of the cofactor. whereas substitution of Met-16 with Thr results in an increase of this pK(a). Also, the pK(a) of Tyr-9 in the absence of substrates was determined directly by fluorescence titration, Substitutions F220Y and M16T resulted in a decrease of 0.5 pK(a) unit and an increase of 0.6 pK(a) unit, respectively, results indicate that a weak hydrogen bond between the engineered Tyr-220 side chain and the aromatic ring face of the catalytic Tyr-9 decreases the pK, of GSH and Tyr-9, and this alters the pH dependence of the enzymatic reaction.
