Temperature Adaptation of Glutathione S-Transferase P1–1

Abstract

Human glutathione S-transferase P1-1 (GST P1-1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35°C, glutathione (GSH) binding to GST P1-1 displays positive cooperativity, whereas negative cooperativity occurs below 25°C. This binding mechanism minimizes changes of GSH affinity for GST P1-1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1-1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding.

Functional Role of the Lock and Key Motif at the Subunit Interface of Glutathione Transferase P1-1

“…Fig. 7, displaying alternative subunit conformations in equilibrium, is supported by studies of Ricci and coworkers (24) suggesting that wild-type GSTP1-1 as a whole somewhat behaves as a thermodynamic system which obeys the Le Chatelier principle. In fact, whenever a physical factor like temperature or viscosity forces the G-site to assume a low affinity conformation for GSH binding, GSTP1-1 opposes this perturbation by developing positive cooperativity and vice versa.…”

“…Mutants G42A, C48S, and K55A display positive cooperativity (21)(22)(23). On the other hand, mutants Y50F (24) and Y50A (the present study) display negative cooperativity of GSH binding. Caccuri et al (24) suggested that Gly 42 , Cys 48 , and Lys 55 point mutations induce cooperativity by distorting the conformation of helix ␣2.…”

“…On the other hand, it is noteworthy that the change of cooperativity caused by a mutation may depend on the position of the mutation rather than on the type of amino acid at this position. For example, negative cooperativity was induced by mutation Y50F (24) and Y50A and positive cooperativity was induced by C48S and C48A (21). Thus, further research will be needed to clarify this phenomenon in GSTP1-1.…”

Thermodynamic Description of the Effect of the Mutation Y49F on Human Glutathione Transferase P1-1 in Binding with Glutathione and the Inhibitor S-Hexylglutathione

“…The Y49F mutant was designed and produced by site-directed mutagenesis, carried out in a two-step polymerase chain reaction (15). Details of this procedure have been reported elsewhere (13). Finally, the Y49F mutant was expressed in TOP 10 Escherichia coli cells and purified as previously reported (14).…”

“…1), could participate in intersubunit communication between active sites of the dimer (1,12). Although some studies have already been performed with the mutant Y49F in the presence of co-substrate (13), there has been no thermodynamic study carried out on this mutant in the absence of the co-substrate.…”