| 61. |
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| 62. |
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| 63. |
- Hederos, Sofia, et al.
(författare)
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A new enzyme by rational design - the incorporation of a single His residue enables efficient thioester hydrolysis by human glutathione transferase A1-1
- 2004
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Ingår i: Proc. Nat. Acad. Sci.. ; 101, s. 13163-13167
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Tidskriftsartikel (refereegranskat)abstract
- A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.
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| 64. |
- Hederos, Sofia, et al.
(författare)
-
Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1.
- 2004
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Ingår i: Proc Natl Acad Sci U S A. - 0027-8424. ; 101:36, s. 13163-7
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Tidskriftsartikel (refereegranskat)abstract
- A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.
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| 65. |
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| 66. |
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| 67. |
- Hegazy, Usama M., et al.
(författare)
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Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies
- 2013
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 425:9, s. 1509-1514
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Tidskriftsartikel (refereegranskat)abstract
- Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kat and 300-fold increased K-M(GSH) values, displays an apparent Hill coefficient of 0.82 +/- 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency k(cat)/K-M(GSH) to near the wild-type value but still yields Hill coefficients of 0.61 +/- 0.08 and 0.86 +/- 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 +/- 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.
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| 68. |
- Hegazy, Usama M., et al.
(författare)
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Modulating Catalytic Activity by Unnatural Amino Acid Residues in a GSH-Binding Loop of GST P1-1
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 376:3, s. 811-826
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Tidskriftsartikel (refereegranskat)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.
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| 69. |
- Hegazy, Usama M., et al.
(författare)
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Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits
- 2006
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 13:9, s. 929-936
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Tidskriftsartikel (refereegranskat)abstract
- Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.
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| 70. |
- Hegazy, Usama M., 1973-
(författare)
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Structure-Function Relationships of Pi Class Glutathione Transferase Studied by Protein Engineering
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr50 in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites.Cys48 and Tyr50 were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr50 with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The KMGSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases kcat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys48 restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.
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