Binding and Kinetic Mechanisms of the Zeta Class Glutathione Transferase

Abstract

The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions. The Zeta class of glutathione transferases (GSTs) has only recently been discovered and hence has been poorly characterized. Here we investigate the substrate binding and kinetic mechanisms of the human Zeta class GSTZ1c-1c by means of pre-steady state and steady-state experiments and site-directed mutagenesis. Binding of GSH occurs at a very low rate compared with that observed for the more recently evolved GSTs (Alpha, Mu, and Pi classes). Moreover, the single step binding mechanism observed in this enzyme is reminiscent of that found for the Theta class enzyme, whereas the Alpha, Mu, and Pi classes have adopted a multistep binding mechanism. Replacement of Cys16 with Ala increases the rate of GSH release from the active site causing a 10-fold decrease of affinity toward GSH. Cys16 also plays a crucial role in co-substrate binding; the mutant enzyme is unable to bind the carcinogenic substrate dichloroacetic acid in the absence of GSH. However, both substrate binding and GSH activation are not rate-limiting in catalysis. A peculiarity of the hGSTZ1c-1c is the half-site activation of bound GSH. This suggests a primitive monomer-monomer interaction that, in the recently diverged GSTP1-1, gives rise to a sophisticated cooperative mechanism that preserves the catalytic efficiency of this GST under stress conditions. Cytosolic glutathione transferases (GSTs, 1The abbreviations used are: GST, glutathione S-transferase; TCHQ, tetrachlorohydroquinone; DCA, dichloroacetic acid. EC 2.5.1.18) are a family of detoxifying enzymes characterized by similar tertiary structures and active site topologies (1Jakoby W.B. Habig W.H. Jakoby W.B. Enzymatic Basis of Detoxification. 2. Academic Press, New York1980: 63-94Google Scholar, 2Armstrong R.N. Chem. Res. Toxicol. 1997; 10: 2-18Crossref PubMed Scopus (963) Google Scholar). GSTs are probably evolved from thioredoxin and glutaredoxin enzymes; in fact, the βαβαββα motif of the N-terminal domain of all cytosolic GSTs superimposes well with the structures of the redox enzymes that are characterized by one or two cysteines in the active site essential for catalysis (3Martin J. Structure (Lond.). 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 4Chivers P.T. Raines R.T. Biochemistry. 1997; 36: 15810-15816Crossref PubMed Scopus (101) Google Scholar, 5Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (109) Google Scholar). A few GSTs still show a cysteine residue in or in close proximity of the active site, and their role has been the object of several studies. In particular, the bacterial GSTB1-1 and the human GSTO1-1 show a GSH molecule in mixed disulfide with the active site cysteine (6Rossjohn J. Polekhina G. Feil S. Allocati N. Masulli M. Di Ilio C. Parker M.W. Structure (Lond.). 1998; 6: 721-734Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Board P.G. Coggan M. Chelvanayagam G. Easteal S. Jermiin L.S. Schulte G.K. Danley D.E. Hoth L.R. Griffor M.C. Kamath A.V. Rosner M.H. Chrunyk B.A. Perregaux D.E. Gabel C.A. Geoghegan K.F. Pandit J. J. Biol. Chem. 2000; 275: 24798-24806Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar). These enzymes, beside the classical GST activity, catalyze thiol-disulfide oxidoreductase reactions, so they could represent a transition structure between an ancestral redox enzyme and a GST-conjugating enzyme (8Caccuri A.M. Antonini G. Allocati N. Di Ilio C. Innocenti F. De Maria F. Parker M.W. Masulli M. Polizio F. Federici G. Ricci G. Biochemistry. 2002; 41: 4686-4693Crossref PubMed Scopus (10) Google Scholar, 9Caccuri A.M. Antonini G. Allocati N. Di Ilio C. De Maria F. Innocenti F. Parker M.W. Masulli M. Lo Bello M. Turella P. Federici G. Ricci G. J. Biol. Chem. 2002; 277: 18777-18784Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In the more recently diverged Alpha, Mu, and Pi class GSTs, the cysteine residues involved in the redox catalysis have been lost. Nevertheless, in GSTP1-1, Cys47 is an important residue lining the G-site. Cys47 is not essential for catalysis of GSTP1-1, but it is highly reactive and forms a peculiar ion pair with Lys54 that is responsible for the high affinity of the enzyme for the substrate GSH. Substitution of Cys47 with Ala decreases the affinity for GSH. In addition this substitution triggers a positive cooperativity for the binding of GSH, and this provided the first indication for a structural intersubunit communication in the GST super-family (10Ricci G. Lo Bello M. Caccuri A.M. Pastore A. Nuccetelli M. Parker M.W. Federici G. J. Biol. Chem. 1995; 270: 1243-1248Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). One of the most recently discovered GSTs is the Zeta class enzyme (GSTZ1-1), which shows very low or no activity toward the classical GST substrates (11Board P.G. Baker R.T. Chelvanayagam G. Jermin L.S. Biochem. J. 1997; 328: 929-935Crossref PubMed Scopus (483) Google Scholar). GSTZ1-1 is characterized by two unusual GSH-dependent reactions as follows: (i) the isomerization of maleylacetoacetate to fumarylacetoacetate, a crucial step in the tyrosine metabolism; (ii) the biotransformation of α-haloacids such as dichloroacetic acid (DCA), a potential carcinogenic contaminant of chlorinated drinking water, which is dehalogenated to glyoxylic acid (12Lantum H.B.M. Board P.G. Anders M.W. Chem. Res. Toxicol. 2002; 15: 957-963Crossref PubMed Scopus (8) Google Scholar, 13Tong Z. Board P.G. Anders M.W. Biochem. J. 1998; 331: 371-374Crossref PubMed Scopus (133) Google Scholar). The Zeta class GST is widely distributed in nature and contains a characteristic motif (SSCX(W/H)RVIAL) in the N-terminal region (11Board P.G. Baker R.T. Chelvanayagam G. Jermin L.S. Biochem. J. 1997; 328: 929-935Crossref PubMed Scopus (483) Google Scholar). Among these residues, the first three (Ser14-Ser15-Cys16) line the active site pocket and have been investigated recently for their possible role in catalysis. In particular, the crystal structure of the human variant GSTZ1a-1a shows the sulfur atom of Cys16 close to the sulfur atom of GSH (14Polekhina G. Board P.G. Blackburn A.C. Parker M.W. Biochemistry. 2001; 40: 1567-1576Crossref PubMed Scopus (103) Google Scholar). It has been shown that C16A mutant is active with different substrates but displays a higher Km value for GSH. Thus, Cys16 seems to play a role in a proper binding of GSH rather than a direct role in catalysis (15Board P.G. Taylor M.C. Coggan M. Parker M.W. Lantum H.B. Anders M.W. Biochem. J. 2003; 374: 731-737Crossref PubMed Scopus (33) Google Scholar). Four polymorphic variants of the human GSTZ1-1 have been detected in the Caucasian population resulting from mutations at positions 32, 42, and 82 (16Blackburn A.C. Tzeng H.-F. Anders M.W. Board P.G. Pharmacogenetics. 2000; 10: 49-57Crossref PubMed Scopus (100) Google Scholar, 17Blackburn A.C. Coggan M. Tzeng H.-F. Lantum H.B. Polekhina G. Parker M.W. Anders M.W. Board P.G. Pharmacogenetics. 2001; 11: 671-678Crossref PubMed Scopus (45) Google Scholar). In the present study, binding and kinetic mechanisms of the most abundant variant, the human GSTZ1c-1c, have been carefully investigated using DCA and GSH as substrates. Moreover, binding and kinetic properties of its C16A mutant have been determined by disclosing multiple roles for the Cys16 residue. Materials—GSH, DCA, NADH; and lactic dehydrogenase were obtained from Sigma; nickel-agarose matrix (nickel-nitrilotriacetic acid) from and of GSTZ1c-1c and C16A GSTZ1c-1c and C16A mutant enzymes were in as and were by nickel-agarose as (16Blackburn A.C. Tzeng H.-F. Anders M.W. Board P.G. Pharmacogenetics. 2000; 10: 49-57Crossref PubMed Scopus (100) Google Scholar). from the at an of The the of the acid as by and Biochem. PubMed Scopus Google and by the acid A of GST used in the W.B. Board P.G. Anders M.W. Chem. Res. Toxicol. 2002; 15: PubMed Scopus Google Scholar). GSH and DCA Binding to of both GSH and DCA to GSTZ1c-1c in in a single with a at different between and at and at The of GSTZ1c-1c for both GSH and DCA were determined by the of the of the the addition of were both for and for and to which the value for the is the observed in the of a of and is the observed at substrate GSH in GSTZ1c-1c and in the C16A of GSH bound to both GSTZ1c-1c and C16A mutant enzymes were obtained with a at or were for the of the enzyme and GSH, and the of GSH at the active site by an The of obtained by of GSH to both GSTZ1c-1c and C16A mutant enzymes in between and the enzyme GSH used in the whereas GSH used at to the low affinity of the mutant enzyme for GSH, GSH used in the region and between and these the of GSH The at with the mutant enzyme obtained under GSH and to GSH by an value for GSH of of the Binding of GSH and DCA to GSTZ1c-1c and C16A kinetic experiments were an kinetic with a Binding of GSH at different between and by of the enzyme in with the of GSH to in The DCA binding at by of the enzyme in with the of DCA to in the Binding of GSH to GSTZ1c-1c and C16A mutant enzymes by the of the at and the of the at to the GSH Binding of DCA to GSTZ1c-1c by the of the at were observed the addition of DCA to the C16A mutant were to a single and kinetic were at different substrates The of substrate to which gives the rate for the of the and the rate for the release of the substrate from the active site The for substrate from Moreover, in the mutant enzyme, the of GSH to a binding to the GSH of the for the Cys16 value for the Cys16 determined from the of the to DCA of both GSTZ1c-1c and C16A mutant enzymes bound to DCA were obtained with a at In a DCA to enzyme in between and were for the of the enzyme and DCA, and the of present at a value from the decrease of the at by an of were to which the value for the Cys16 with DCA as activity at and using a Blackburn A.C. Board P.G. Anders M.W. Chem. Res. Toxicol. 2000; PubMed Scopus Google Scholar). The GSH with the C16A DCA with the C16A of and catalytic of GSTZ1c-1c or C16A mutant The determined under the in the absence of The of the used as the enzyme, to of the One of activity is as the of enzyme that of DCA at The of activity determined in between and in the of substrate for the enzyme and for the C16A mutant with the of the GSH were and for the and the C16A mutant enzymes, kinetic with both GSTZ1c-1c and C16A mutant enzymes at and by both GSH and DCA from with the C16A to with the C16A a matrix of substrate The of of the substrate to a binding which and for the substrate at co-substrate were as to the steady-state kinetic mechanism. In the enzyme, of and of the of the co-substrate the affinity of both DCA and GSH toward the enzyme and toward the enzyme with the co-substrate Biochemistry. 2000; PubMed Scopus Google Scholar). In the C16A of for DCA the of the GSH the affinity of GSH toward the enzyme and the affinity of DCA toward the enzyme in with GSH. Moreover, a of GSH is the the of addition of substrates to the enzyme Biochemistry. 2000; PubMed Scopus Google Scholar). Binding of GSH and DCA to binding of both GSH and DCA to GSTZ1c-1c at different between and and at by the of the of the at different of substrates. were to and of and were found at and a of the for GSH the Binding experiments with the substrate DCA a of at which is with the value obtained for GSH at the in the C16A only a of the observed addition of GSH, whereas DCA not Thus, in the mutant enzyme, binding of substrates not by using this GSH in GSTZ1c-1c and in C16A crystal structure of that the of the GSH is the N-terminal of (14Polekhina G. Board P.G. Blackburn A.C. Parker M.W. Biochemistry. 2001; 40: 1567-1576Crossref PubMed Scopus (103) Google Scholar). it has been that the proximity of a to the positive of a decrease the of the as for the residue in GSTs (15Board P.G. Taylor M.C. Coggan M. Parker M.W. Lantum H.B. Anders M.W. Biochem. J. 2003; 374: 731-737Crossref PubMed Scopus (33) Google Scholar). that occurs in the Zeta class from of bound to GSTZ1c-1c or C16A mutant enzymes were between and and show an at which is of the ion A and at different under GSH that the of the bound GSH in the C16A mutant is with that of the enzyme, and both are and Moreover, at both and the of GSH not active site both in the and also stress that of C16A mutant is obtained only at GSH. In fact, at and GSH, the of GSH is of GSH kinetic experiments were to the binding mechanism of GSH to both GSTZ1c-1c and C16A mutant of the binding of GSH by the of the of the at to GSH binding and the at to the GSH Moreover, the to the binding to the mutant enzyme, which only a were at and and and in are shown the obtained at The rate for both the and the a GSH to This suggests that GSH forms a by a interaction both in the and in C16A mutant In GSTZ1c-1c, the is with the at and whereas at the GSH binding and the as two kinetic In the C16A this is observed at both and with the of step with the GSH binding The kinetic and for the binding of GSH were from by of the substrate were from the These with obtained from steady-state that are by and a decrease of affinity for GSH by Cys16 both at and for GSH and DCA of GSH to of DCA to of GSH to C16A in a substitution of Cys16 with Ala an of both and for GSH However, value increases whereas a has been observed for the Thus, a release of GSH from the seems to the of the affinity toward GSH in the C16A mutant and suggests a role for Cys16 in the of an high affinity active site structure by the of its potential of DCA kinetic experiments were to the binding mechanism of DCA to both GSTZ1c-1c and C16A mutant of the binding of DCA by the of the of the at to DCA and binding experiments with DCA by the of a interaction with the of Cys16 as observed W.B. Board P.G. Anders M.W. Chem. Res. Toxicol. 2002; 15: PubMed Scopus Google Scholar). this in the of the present experiments we not interaction with the enzyme as by the of the enzyme activity at the of the experiments not The rate for the a DCA to and gives and a value of at and and The kinetic and found for DCA are to the found for the binding of GSH, that GSTZ1c-1c and both substrates at of the co-substrate binding only in the enzyme no were observed the addition of DCA to the C16A mutant of Cys16 is by DCA under conditions. The one Cys16 and a enzyme W.B. Board P.G. Anders M.W. Chem. Res. Toxicol. 2002; 15: PubMed Scopus Google Scholar). This only occurs in the of GSH as shown by Tzeng and Blackburn A.C. Board P.G. Anders M.W. Chem. Res. Toxicol. 2000; PubMed Scopus Google Scholar). In an to the interaction of the enzyme with DCA, we the to the co-substrate DCA an of a at A very similar observed of the enzyme with the acid not However, in the of DCA, a of Cys16 not to the absence of GSH in the and by the of the enzyme activity at the of the observed DCA to the C16A mutant binding of DCA probably the of the Cys16 its in the The is to the of the present at and its a value of a value than that of the cysteine in an at close to that of the of GSH the value of the Cys16 at for active The present that only one Cys16 shows and this the half-site of this residue observed W.B. Board P.G. Anders M.W. Chem. Res. Toxicol. 2002; 15: PubMed Scopus Google Scholar). of Cys16 DCA binding observed by of to the co-substrate binding of the between DCA and a few used to the kinetic mechanism of the of this enzyme acid (15Board P.G. Taylor M.C. Coggan M. Parker M.W. Lantum H.B. Anders M.W. Biochem. J. 2003; 374: 731-737Crossref PubMed Scopus (33) Google of the catalytic mechanism of GSTZ1c-1c with DCA is of this carcinogenic molecule is a contaminant of chlorinated drinking Thus, we a to the rate of glyoxylic acid of this molecule by the Biochemistry. 2000; PubMed Scopus Google Scholar). The of the at and at is for the enzyme and for the C16A in the and in the C16A mutant enzymes, the is and the of the GSH and This suggests that the rate-limiting step the the GSH activation and that of Cys16 not the rate-limiting step of The of the rate obtained at and by both DCA and GSH, with both the and the C16A The family of for the GSH is to that obtained for DCA in the enzyme A and and this is with a mechanism in which DCA and GSH bind to the In this of and of the of the co-substrate the affinity of both DCA and GSH toward the enzyme and toward the enzyme with the co-substrate New Scholar). The of GSH and DCA from the and are and The of GSH and DCA from the are and these the is to that the affinity toward one substrate is by the binding of the co-substrate to the A catalytic mechanism for GSTZ1c-1c is in kinetic mechanism for the DCA by of Cys16 a in the kinetic in the C16A the family of for DCA the and this is of a mechanism in which GSH first In this of for DCA the different the affinity of GSH toward the enzyme and the affinity of DCA toward the enzyme in with GSH Moreover, a of GSH is the substrate is and at the DCA is This is a indication of an mechanism with GSH to the enzyme DCA New Scholar). A catalytic mechanism for C16A mutant is in kinetic mechanism for the DCA by C16A of GSTZ1c-1c has been and the role of the Cys16 residue in the biotransformation of DCA has also been The Zeta class GSTs from a of a motif in the N-terminal region that the two residues found in the active site of the enzyme and This enzyme shows a site similar to found in GSTs, and it has been to to the Zeta class GST Biochemistry. 2000; PubMed Scopus Google Scholar). in a in which the first one the of GSH the and the step the of the residue. In the by GSTZ1c-1c with DCA, the first step the of GSH the but Cys16 to in not to essential for the catalytic Among GSTs, the of a cysteine residue in the active site has been observed in the and and both cysteine residues are close to the of Cys16 in GSTZ1-1 active site (14Polekhina G. Board P.G. Blackburn A.C. Parker M.W. Biochemistry. 2001; 40: 1567-1576Crossref PubMed Scopus (103) Google Scholar). The and class GSTs are characterized by a GSH molecule in mixed disulfide with the active site cysteine and catalyze reactions P.G. Coggan M. Chelvanayagam G. Easteal S. Jermiin L.S. Schulte G.K. Danley D.E. Hoth L.R. Griffor M.C. Kamath A.V. Rosner M.H. Chrunyk B.A. Perregaux D.E. Gabel C.A. Geoghegan K.F. Pandit J. J. Biol. Chem. 2000; 275: 24798-24806Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 9Caccuri A.M. Antonini G. Allocati N. Di Ilio C. De Maria F. Innocenti F. Parker M.W. Masulli M. Lo Bello M. Turella P. Federici G. Ricci G. J. Biol. Chem. 2002; 277: 18777-18784Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In particular, the residue in the class GST is essential for this and by the The crystal structure of the human Zeta class GST shows that Cys16 is in close proximity to the sulfur atom of the bound GSH (14Polekhina G. Board P.G. Blackburn A.C. Parker M.W. Biochemistry. 2001; 40: 1567-1576Crossref PubMed Scopus (103) Google but the enzyme is not to catalyze redox reactions with all these it is that the active site cysteine in this enzyme is no a catalytic residue. However, the Cys16 is characterized by the very low value of than that of cysteine in in Pi class GST Cys47 is characterized by a very low value (10Ricci G. Lo Bello M. Caccuri A.M. Pastore A. Nuccetelli M. Parker M.W. Federici G. J. Biol. Chem. 1995; 270: 1243-1248Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In this the of Cys47 is in an ion pair with the of and this interaction the structural of the resulting in an affinity for GSH. in the GSTZ1c-1c, Cys16 is in the at and this by the of a residue in the G-site. the active site, compared with GSTs, is characterized by high and the positive of with from the of by the Cys16 in the absence of the The present that substitution of Cys16 with has multiple the binding and kinetic mechanisms of the Zeta class GST, the of this residue. Binding of GSH to the enzyme occurs at a very low rate compared with that observed for the more recently diverged Alpha, Mu, and Pi class GSTs A.M. Antonini G. Board P.G. Parker M.W. M. Lo Bello M. Federici G. Ricci G. Biochem. J. PubMed Scopus Google Scholar). In these enzymes the rate of the GSH binding at are of higher than found for the Zeta enzyme at The and observed for the Zeta class GST are similar to obtained for the Theta class and at A.M. Antonini G. Board P.G. J. Parker M.W. Turella P. Federici G. Lo Bello M. Ricci G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). in the Theta enzyme the is by the of the whereas in the Zeta class GST, GSH is in a very between the two the single step binding mechanism observed in the Zeta enzyme is also found in the Theta enzyme, whereas the more recently evolved GSTs have adopted a multistep binding mechanism A.M. Antonini G. Board P.G. Parker M.W. M. Lo Bello M. Federici G. Ricci G. Biochem. J. PubMed Scopus Google Scholar). of Cys16 increases the rate of GSH release from the active site the 10-fold decrease of affinity toward GSH at Moreover, the mutant enzyme shows very low affinity toward GSH in the at GSH binding and activation of GSH are in this GSH, the of the mutant enzyme is higher than that of the enzyme in the that not the binding of GSH and probably increases the active site A more of is observed the binding of the co-substrate The C16A mutant enzyme has the to bind DCA, and binding occurs only in the enzyme bound to GSH. the steady-state of the between DCA and GSH is also by the and from a mechanism in which DCA and GSH bind to the enzyme to an mechanism in which GSH The crystal structure of is a with GSH and a ion from the The of Cys16 forms direct with the of and and with the GSH of as well as with the GSH In the GSH the of and it also with the ion that is to the binding of In the ion direct with to a residue in DCA the active site studies. the of GSH to play an important with in the binding of DCA (14Polekhina G. Board P.G. Blackburn A.C. Parker M.W. Biochemistry. 2001; 40: 1567-1576Crossref PubMed Scopus (103) Google Scholar). the of Cys16 the of GSH to binding of DCA in the It is that the of the GSH bound to hGSTZ1c-1c not active site under GSH The half-site GSH activation by a cooperativity the two enzyme only one to the GSH This is reminiscent of the Theta class in which GSH is but only of the enzyme population is A.M. Antonini G. Board P.G. J. Parker M.W. Turella P. Federici G. Lo Bello M. Ricci G. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of a cooperative half-site in the GST is a of GST it of the evolved GSTs that a residue to the GSH whereas in the recently diverged GSTs, which a tyrosine residue for GSH it is a more sophisticated mechanism of as observed in and most in the and classes as is to bind and GSH both but cooperativity to the first is by or G. Caccuri A.M. Lo Bello M. Parker M.W. Nuccetelli M. Turella P. Di Federici G. Biochem. J. 2003; PubMed Scopus (33) Google Scholar). In this enzyme shows a of substrate which for the of very high or low GSH In this enzyme, one displays a affinity for GSH with a positive value of whereas binding of GSH to the is with a value A.M. G. P. M. Nuccetelli M. Federici G. Lo Bello M. Ricci G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In GSTZ1c-1c, binding of GSH to the first shows an of a value similar to that found for GSTP1-1, but a is observed in the of the In the Zeta class GST the it is not to GSH, and this an to bind GSH. This primitive cooperativity could the for a of structural of the enzyme, whereas in GSTP1-1, it seems to evolved toward a mechanism that the of or by the catalytic efficiency of this