Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies
2013 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, no 9, 1509-1514 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2013. Vol. 425, no 9, 1509-1514 p.
allostery, cooperativity, ethacrynic acid, glutathione transferase, unnatural amino acid
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:su:diva-91038DOI: 10.1016/j.jmb.2013.01.038ISI: 000318585800013OAI: oai:DiVA.org:su-91038DiVA: diva2:630498
FunderSwedish Research CouncilSwedish Cancer Society