1usb
From Proteopedia
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==Overview== | ==Overview== | ||
| - | 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, ... | + | 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. |
==About this Structure== | ==About this Structure== | ||
| - | 1USB is a | + | 1USB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with CL, K and GSH as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Glutathione_transferase Glutathione transferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.5.1.18 2.5.1.18] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1USB OCA]. |
==Reference== | ==Reference== | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 15:04:57 2007'' |
Revision as of 12:59, 5 November 2007
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RATIONAL DESIGN OF A NOVEL ENZYME- EFFICIENT THIOESTER HYDROLYSIS ENABLED BY THE INCORPORATION OF A SINGLE HIS RESIDUE INTO HUMAN GLUTATHIONE TRANSFERASE A1-1
Overview
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.
About this Structure
1USB is a Single protein structure of sequence from Homo sapiens with CL, K and GSH as ligands. Active as Glutathione transferase, with EC number 2.5.1.18 Structure known Active Site: AC1. Full crystallographic information is available from OCA.
Reference
Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1., Hederos S, Broo KS, Jakobsson E, Kleywegt GJ, Mannervik B, Baltzer L, Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13163-7. Epub 2004 Aug 27. PMID:15333749
Page seeded by OCA on Mon Nov 5 15:04:57 2007
Categories: Glutathione transferase | Homo sapiens | Single protein | Jakobsson, E. | Kleywegt, G.J. | CL | GSH | K | Glutathione | Transferase
