The catalytic mechanism of glutathione S-transferase (GST). Spectroscopic determination of the pKa of Tyr-9 in rat alpha 1-1 GST.
The rat alpha 1-1 glutathione S-transferase (GST) contains a single, non-essential tryptophan and only 8 tyrosines in each subunit. One of these tyrosines, Tyr-9, hydrogen bonds to the substrate glutathione and stabilizes the nucleophilic thiolate anion. Two mutant proteins that allow for the spectrocopic determination of the pKa of this catalytic residue have been constructed. The W21F mutant provides a fully active GST with no tryptophans, and the double mutant W21F/Y9F lacks both tryptophan and the active site tyrosine. The intrinsic fluorescence and absorbance properties of these mutants are dominated by tyrosine. Fluorescence emission, fluorescence excitation, and absorbance spectral changes of samples containing the W21F mutant at several pH values in the range 6.8-9.0 reveal a pH-dependent increase in the contribution of tyrosinate. No spectral changes are observed with the W21F/Y9F protein in this pH range. At pH 12.5, both proteins exhibit complete deprotonation of all tyrosines. The pKa of Tyr-9 determined from these spectroscopic changes is 8.3-8.5. The changes in absorbance at 250 and 295 nm correspond to titration of 0.95 +/- 0.29 tyrosines/subunit in the W21F protein between pH 6.9 and 9.3. Moreover, addition of the inhibitor S-hexylglutathione results in an apparent increase in the pKa of Tyr-9. Together, these results indicate that the catalytically active Tyr of GSTs has a pKa value that is 1.8-2.0 pKa units below tyrosine in solution. It is likely that this decrease in the pKa of Tyr-9 contributes to catalysis by altering the equilibrium position of the proton shared between Tyr-9 and GSH, and this active site residue may function as a general base catalyst in addition to a hydrogen bond donor.
Atkins
W M
WM
Department of Medical Chemistry, School of Pharmacy, University of Washington, Seattle 98195.
Wang
R W
RW
Bird
A W
AW
Newton
D J
DJ
Lu
A Y
AY
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004735:Energy Transfer; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine
D011994:Recombinant Proteins; D014443:Tyrosine; D005982:Glutathione Transferase