Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase

Glutathione transferases are detoxification enzymes that catalyze the addit ion of glutathione (GSH) to a wide variety of hydrophobic compounds. Althou gh this group of enzymes has been extensively characterized by crystallogra phic studies, Little is known about their dynamic properties. This study in vestigates the role of protein dynamics in the mechanism of a human class m u enzyme (GSTM2-2) by characterizing the motional properties of the unligan ded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex, [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these liga nds are 10-20-foId lower than the diffusion limit, suggesting dynamic confo rmational heterogeneity of the active site. The off-rate of GSDNB is simila r to the turnover number for its enzymatic formation, suggesting that produ ct release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substra te. The dynamic properties of GSTM2-2 were investigated over a wide range o f time scales using N-15 nuclear spin relaxation, residual dipolar coupling s, and amide hydrogen-deuterium exchange rates. These data show that the ma jority of the protein backbone is rigid on the nanosecond to picosecond tim e scale for all forms of the enzyme. The presence of motion on the millisec ond to microsecond time scale was detected for a small number of residues w ithin the active site. These motions are Likely to play a role in facilitat ing substrate binding and product release. The residual dipolar couplings a lso show that the conformation of the active site region is more open in so lution than in the crystalline environment, further enhancing Ligand access ibility to the active site. Amide hydrogen-deuterium exchange rates indicat e a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate o f a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site regi on. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 comp lex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic resid ues in the active site, including Met108. Replacement of Met108 with Ala in creases the turnover number of the enzyme by a factor of 1.7.