Rational Modulation of the Catalytic Activity of A1-1 Glutathione S-Transferase:  Evidence for Incorporation of an On-Face (π···HO−Ar) Hydrogen Bond at Tyrosine-9

• Published in: Biochemistry (Easton) 1996-09, Vol.35 (37), p.11938-11944
• Main Authors: Dietze, Eric C, Ibarra, Catherine, Dabrowski, Michael J, Bird, Andrew, Atkins, William M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Crystallography, X-Ray; DNA Primers; Glutathione Transferase - chemistry; Glutathione Transferase - metabolism; Hydrogen Bonding; Hydrogen-Ion Concentration; Isoenzymes - chemistry; Isoenzymes - metabolism; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Conformation; Rats; Spectrometry, Fluorescence; Tyrosine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi961073r

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 are 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 (π···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·GSH 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. Together, these 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 a of GSH and Tyr-9, and this alters the pH dependence of the enzymatic reaction.