Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of Substrate Binding to the Active Site of Glutathione Transferases

Abstract

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK a ‫؍‬ 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k on and k off values at least hundred times lower (k on ‫؍‬ (2.7 ؎ 0.8) ؋ 10 4 M ؊1 s ؊1 , k off ‫؍‬ 36 ؎ 9 s ؊1 , at 37°C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK a value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.

Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

“…The best fit of these kinetic data to the minimal Scheme II (see "Experimental Procedures") yields apparent microscopic rate constants reported in Table I. It appears that k on and k off are close to those previously found for the formation of the binary complex E-GSH (10). Thus, the presence of Msu does not affect the binding of GSH, and this co-substrate interacts with the active site at a higher or similar velocity compared with GSH.…”

“…pK a of GSH in the binary complex with this mutant (10). Thus, the replacement of Arg-107 yields a similar shift of pK a both in the binary and in the ternary complexes of hGSTT2-2.…”

“…1B). The best fit of the experimental data to Equation 1 yields an apparent pK a1 ϭ 5.6 Ϯ 0.20 and pK a2 ϭ 9.2 Ϯ 0.20. pK a1 is close to the apparent pK a of the bound GSH (pK a ϭ 6.1), as calculated in the binary complex (10). The presence of Msu in the active site probably causes a more favorable deprotonation of the bound GSH.…”

“…Hill plots, obtained from experiments at various pH values, yield a Hill coefficient ranging from 0.7 to 0.8. This non-Michaelian kinetic behavior is unexpected as the isothermal binding of GSH has been found to be hyperbolic (10). Thus, this may correspond to a true cooperativity only if the presence of the co-substrate in the active site causes an intersubunit communication that is absent without it.…”

“…One crucial difference resides in the structural rigidity of the hGSTT2-2 that may be the origin of most of the kinetic oddities described above. A comparison of the mobility profile of representative GST isoenzymes has been presented in the accompanying paper (10). This comparison demonstrates the relative rigidity of the primordial enzyme compared with the more recently evolved GSTs and, in particular, the absence of a sequence of "cold" and "hot" regions along the polypeptide chain.…”