2J9H

Crystal structure of human glutathione-S-transferase P1-1 cys-free mutant in complex with S-hexylglutathione at 2.4 A resolution

Summary for 2J9H

• Entry DOI: 10.2210/pdb2j9h/pdb
• Related: 10GS 11GS 12GS 13GS 14GS 16GS 17GS 18GS 19GS 1AQV 1AQW 1AQX 1EOG 1EOH 1GSS 1KBN 1LBK 1MD3 1MD4 1PGT 1PX6 1PX7 1ZGN 20GS 21GS 22GS 2A2R 2A2S 2GSS 2PGT 3GSS 3PGT 4GSS 4PGT 5GSS 6GSS 7GSS 8GSS 9GSS
• Descriptor: GLUTATHIONE S-TRANSFERASE P, S-HEXYLGLUTATHIONE (3 entities in total)
• Functional Keywords: p1-1, transferase, glutathione, polymorphism, glutathione transferase
• Biological source: HOMO SAPIENS (HUMAN)
• Total number of polymer chains: 2
• Total formula weight: 47085.60

Authors

Tars, K.,Hegazy, U.M.,Hellman, U.,Mannervik, B. (deposition date: 2006-11-08, release date: 2006-11-14, Last modification date: 2023-12-13)

Primary citation

Hegazy, U.M.,Tars, K.,Hellman, U.,Mannervik, B.
Modulating Catalytic Activity by Unnatural Amino Acid Residues in a Gsh-Binding Loop of Gst P1-1.
J.Mol.Biol., 376:811-, 2008

PubMed Abstract: The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.

PubMed: 18177897
DOI: 10.1016/J.JMB.2007.12.013

Experimental method

X-RAY DIFFRACTION (2.4 Å)