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J Mol Biol. 2013 May 13;425(9):1509-14. doi: 10.1016/j.jmb.2013.01.038. Epub 2013 Feb 8.

Hidden allostery in human glutathione transferase p1-1 unveiled by unnatural amino acid substitutions and inhibition studies.

Author information

1
Molecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, 12311 Cairo, Egypt.

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 kcat and 300-fold increased KM(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 kcat/KM(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.

PMID:
23399543
DOI:
10.1016/j.jmb.2013.01.038
[Indexed for MEDLINE]

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