Usama M. Hegazy

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

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr50 in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr50 to Ala. Although the key residue is located far from the catalytic center, the kcat value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the kcat value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr50, thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix α2, which interacts directly with GSH.

Mapping of Amino Acid Substitutions Conferring Herbicide Resistance in Wheat Glutathione Transferase

We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates. The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides. Predictive quantitative models like those presented here have broad applicability for bioengineering.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

OBJECTIVE Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. METHODS We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. RESULTS We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. CONCLUSION AND PRACTICAL IMPLICATIONS In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies

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 kcat and 300-fold increased KM(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 kcat/KM(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.

Modulating catalytic activity by unnatural amino acid residues in a GSH-binding loop of GST P1-1.

The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.

Identification of new inhibitors for human hematopoietic prostaglandin D2 synthase among FDA-approved drugs and other compounds.

OBJECTIVE Hematopoietic prostaglandin D2 synthase (HPGDS) is a member of the Sigma class glutathione transferases (GSTs) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2, a mediator of allergy and inflammation responses. Selective inhibitors of human HPGDS are expected to be of therapeutic importance in relieving symptoms related to allergy and asthma. Hence, a collection of diverse FDA-approved compounds was screened for potential novel applications as inhibitors of HPGDS. METHODS The catalytic activity of purified HPGDS was used for inhibition studies in vitro. RESULTS Our inhibition studies revealed 23 compounds as effective inhibitors of HPGDS with IC50 values in the low micromolar range. Erythrosine sodium, suramin, tannic acid and sanguinarine sulfate were characterized with IC50 values of 0.2, 0.3, 0.4, and 0.6 μM, respectively. Kinetic inhibition analysis showed that erythrosine sodium is a nonlinear competitive inhibitor of HPGDS, while suramin, tannic acid and sanguinarine sulfate are linear competitive inhibitors. CONCLUSION The results show that certain FDA-approved compounds may have pharmacological effects not previously realized that warrant further consideration in their clinical use.