©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Human Glutathione Transferase P1-1
MUTATION OF Cys-47 INDUCES A POSITIVE COOPERATIVITY IN GLUTATHIONE TRANSFERASE P1-1 (*)

(Received for publication, August 10, 1994; and in revised form, October 31, 1994)

Giorgio Ricci (1)(§) Mario Lo Bello (1) Anna Maria Caccuri (1) Anna Pastore (1) Marzia Nuccetelli (1) Michael W. Parker (2)(¶) G. Federici (1)

From the  (1)Department of Biology, University of Rome ``Tor Vergata,'' 00133 Rome, Italy and the (2)St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUDING REMARKS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT


Glutathione transferase P1-1 (EC 2.5.1.18) is a dimeric enzyme composed of identical subunits each containing one binding site for GSH and a second for the co-substrate e.g. 1-chloro-2,4-dinitrobenzene. Steady-state kinetics are strictly hyperbolic toward both these substrates. Replacement of Cys-47 with alanine or serine decreases the affinity for GSH and triggers a positive kinetic cooperativity with respect to this substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101A and C47S/C101S double mutants display lower affinity for GSH and higher Hill coefficients (1.57 and 1.56, respectively) when compared with C47A and C47S single mutants. Conversely, replacement of Cys-101 with alanine or serine does not yield any cooperativity and any marked change of kinetic parameters. Fluorometric experiments gave sigmoidal isothermic GSH binding curves for all the Cys-47 mutants, with Hill coefficients similar to that obtained by the kinetic approach. These data, together with the activation experiments performed in the presence of S-hexylglutathione, suggest that the substitution of Cys-47 yields a dimeric low-affinity enzyme which can be converted to a higher affinity state upon binding of GSH on one subunit. These findings indicate a structural communication between subunits which may be revealed by the lack of a peculiar electrostatic bond between the thiolate form of Cys-47 and the protonated amino group of Lys-54.


INTRODUCTION


The glutathione transferases (EC 2.5.1.18) (GST) (^1)are a family of detoxicating enzymes able to conjugate the sulfhydryl function of GSH to a large number of electrophilic compounds(1, 2, 3, 4, 5, 6) . The cytosolic GSTs, which have been grouped into four gene-independent classes, Alpha, Pi, Mu(7) , and Theta(8) , are homodimeric or heterodimeric proteins composed of subunits of about 25 kDa. Crystal structures of homodimeric GSTs from thee gene classes (Alpha, Pi, and Mu) have been recently solved (9, 10, 11, 12) and provide useful data for elucidating the catalytic mechanism. From these structural studies it is also evident that all these proteins have very similar tertiary folds. Each subunit can be divided in two domains: a smaller amino-terminal domain (Domain I) which contains the binding site for GSH (G-site), and a larger carboxyl-terminal domain (Domain II) which binds the co-substrate in an hydrophobic cavity or cleft (H-site). Crystallographic data suggest that, in the dimeric protein, the two G-sites are structurally independent as well as the two H-sites. The kinetic independence of the subunits of GSTs has been well established in studies of a number of rat isoenzymes composed of four different subunits(13) ; furthermore, steady-state kinetic pathways of the human placenta GST (class Pi), as well as of the rat liver GST 1-1 (class Alpha), are fully Michaelian and consistent with a rapid equilibrium random sequential Bi Bi mechanism(14, 15) . Other isoenzymes such as the rat GST 3-3 and 3-4 (class Mu) and GST 2-2 (class Alpha) exhibit non-Michaelian kinetic behavior which would suggest negative cooperativity, but this pathway has been well fitted with the simplest steady-state random Bi Bi mechanism(16) , also on the basis of the strictly hyperbolic binding isotherms of substrates and products(17) . Thus, it is generally accepted that GSTs lack kinetic cooperativity or regulatory behavior. In this paper we report that replacement of a single residue in the Pi isoenzyme triggers a marked kinetic and ligand binding cooperativity.

During investigations about possible structural roles of Cys-47 and other sulfhydryls in the GST P1-1, we constructed six mutants by different substitutions of Cys-47 and Cys-101, the most reactive cysteines among the four present in each subunit. C47A and C101A mutants of the rat GST P and C47S, C101S mutants of the human GST P1-1 have been previously studied by two different research groups(18, 19) . The kinetic parameters of these mutated enzymes were found approximately similar to those of the wild type but the substitution of Cys-47 lowers the affinity for GSH considerably. No anomalous kinetic behavior has been observed. In this paper we demonstrate that some of these mutated enzymes display positive cooperativity. In particular, the replacement of Cys-47 is crucial in this phenomenon: C47A and C47S mutants and C47A/C101A and C47S/C101S double mutants give similar sigmoidal dependence of the reaction velocity on GSH concentration and homotropic behavior on GSH binding, whereas the C101A and C101S mutants as well as the wild type are noncooperative enzymes.

Cys-47, a conserved residue in the Pi class isoenzymes, has been the object of many investigations as it is highly reactive and its chemical modification inactivates the enzyme possibly by inducing unfavorable conformational changes for GSH binding or by blocking a structural transition during catalysis(11) . It is not a structural part of the active site but it is placed close the G-site(11) . Previously, we demonstrated that it may form an intrasubunit disulfide link with Cys-101 (20) leading to an inactive enzyme form. Its sulfhydryl group has also a very low pK value, and it is probably linked in ion pair with the protonated group of Lys-54(21) . Results reported here confirm the key role of this residue in the activity modulation of GST P1-1.


EXPERIMENTAL PROCEDURES


Preparation of the C47A, C47S, C101A, C101S, C47A/C101A, and C47A/C101A Mutants

Construction of mutants, purification, and check of purity are described in the subsequent paper in this issue (22) .

Spectrophotometric Measurements

GST activity was assayed spectrophotometrically at 340 nm essentially as described by Habig et al.(23) . Spectrophotometric measurements were performed in a double beam Uvicon 940 spectrophotometer (Kontron Instruments) equipped with a thermostatted cuvette compartment. Initial rates were measured at 0.1-s intervals for a total period of 12 s after a lag time of 5 s. Enzymatic rates were corrected for the spontaneous reaction. Standard incubation mixture contained, in order of addition, 0.1 M potassium phosphate buffer, pH 6.5, 1 mM EDTA, 1 mM GSH, 1 mM CDNB, and about 1 µg of GST. Stock solutions of CDNB dissolved in absolute ethanol were used so the final concentration of ethanol in the reaction mixture was constant at 5% (v/v). Stock solutions of GSH (100 mM and 10 mM) were prepared in 0.1 M potassium phosphate buffer, pH 6.5, and 1 mM EDTA; the pH was adjusted to 6.5 by adding 2 M KOH. GSH solutions were kept under nitrogen in an ice bath to prevent oxidation.

Kinetic Analysis

The dependence of initial rate upon GSH concentration was followed by varying GSH from 0.025 mM to 20 mM (about 16 experimental points) by keeping constant the CDNB concentration at 1 mM. Kinetic data were analyzed by the GraphPAD InPlot (version 3.1) (GraphPAD Software, San Diego, CA) or by the KaleidaGraph (version 2.0.2) (Abelbeck Software) computer programs and were fitted: (i) by nonlinear least squares regression analysis with rate equations expressing positive cooperativity between the two GSH binding sites (as follows).


From this equation a reliable K(m) value cannot be evaluated because the interaction factor alpha and K(m) are not independent variables.


A sigmoidal curve is obtained from by plotting v versus log [S]. The best fit fulfills V(max), [S](0.5), and the Hill coefficient (n(H)); (ii) by linear least squares regression analysis with a linear rate equation.


In this analysis, V(max) value was obtained by fitting with .

The dependence of the enzymatic velocity upon CDNB concentration was studied at a constant GSH concentration of 1 mM or 0.5 mM and varying the CDNB concentration from 0.05 mM to 2 mM. Data were analyzed as above or by using Lineweaver-Burk double-reciprocal plots.

Kinetic parameters reported in this paper represent the mean of at least four different experimental sets.

Fluorometric Measurements

The intrinsic fluorescence of GST P1-1 was measured with a Perkin-Elmer LS-5 fluorometer equipped with a thermostatted sample holder set at 25 °C. Excitation was at 280 ± 2.5 nm, and the emission was at 340 ± 2.5 nm. A number of samples containing 2 µM GST (wild type or mutants) in 2 ml of 0.1 M potassium phosphate buffer, pH 6.5, were prepared and the fluorescence intensity measured. A suitable amount of GSH, dissolved in the same buffer, was then added to each sample and the fluorescence intensity measured after mixing. The binding of GSH to the mutant GSTs quenches the intrinsic fluorescence of the enzyme as previously reported for the wild type GST P1-1(24) . The experimental data, corrected both for dilution and for inner filter effects, were fitted with and (see above) by substituting DeltaF(c) (observed fluorescence quenching at fixed ligand concentration) and DeltaF (fluorescence quenching at infinite ligand concentration) for v and V(max), respectively.

Activation by S-Hexylglutathione

Activation experiments were performed at 25 °C at fixed CDNB concentration (1 mM) and at about 0.1 [S](0.5) GSH concentration in the presence of variable amounts of S-hexylglutathione. The reaction was started by the addition 1 µg of GST.


RESULTS AND DISCUSSION


Kinetic Properties of C47A, C47S, C101A, C101S, C47A/C101A, and C47S/C101S Mutants

The substitution of Cys-47 by alanine or serine in the GST P1-1 does not affect remarkably the V(max) value or the affinity toward CDNB, whereas the affinity for GSH is lowered (Table 1). When compared with the wild type, C47A and C47S mutants display a GSH concentration that gives half the maximal velocity, [S](0.5), about six and eight times higher, respectively. These results are somewhat in agreement with that previously reported for the C47S mutant of the human isoenzyme(19) . In respect with these single mutants, C47A/C101A and C47S/C101S double mutants exhibit slightly increased V(max) and higher [S](0.5) values. On the contrary, the kinetic parameters of C101A and C101S mutants are similar to that of wild type. These kinetic data confirm the nonessential character of Cys-47 in the catalytic mechanism but suggest a role of this residue in the maintenance of a proper structure of the G-site. By considering the structural similarity between cysteine with serine, the lowered affinity for GSH of the C47S mutant should be related to the replacement of the dissociable sulfhydryl group with the undissociable hydroxyl group. In other words, the peculiar ion pair, which has been suggested to occur in the wild type structure between the thiolate form of Cys-47 and the protonated amino group of Lys-54 (21) , cannot exist in the C47A and C47S mutants, as well as in the double mutants. The absence of this electrostatic bond may be the origin of a structural perturbation of the G-site. A more unfavorable conformation seems to occur in the double mutants C47A/C101A and C47S/C101S (see Table 1), thereby suggesting some, but not crucial, role of Cys-101 in the stabilization of the G-site proper structure. Surprisingly, the increased V(max) of the double mutants also indicates that the substitution of Cys-101 lowers the energy barrier of the rate-limiting step. The reason of this finding is unclear, at present.




Cooperative Behavior upon Replacement of Cys-47

When the concentration of GSH was varied from 0.05 mM to 20 mM at fixed nonsaturating CDNB concentration (1 mM), the steady-state kinetics of the C47A, C47S, C47A/C101A, and C47S/C101S mutants do not obey the usual hyperbolic rate equation. The data were well fitted to a rate equation expressing positive cooperativity between the two GSH binding sites (see under ``Experimental Procedures''). As an example, the GSH saturation curve for the C47S mutant is given in Fig. 1and compared with the theoretical pattern expected for a noncooperative GST with the same V(max) and [S](0.5) values. The deviation from the Michaelian kinetics is also evident by the convex curve obtained in the classical double-reciprocal plot 1/vversus 1/[S] (Fig. 2a). The extent of a positive cooperativity is usually measured in terms of Hill coefficient (n(H)); for a dimeric enzyme with two interacting sites, it ranges from 2 (infinite cooperativity) to 1 (no cooperativity). A summary of the Hill coefficients for all mutants and for the wild-type is given in Table 2. n(H) values range from 1.43 to 1.31 for the single mutants of Cys-47 and from 1.57 to 1.56 for the double mutants. Conversely, the wild type enzyme, the C101A and C101S mutants follow the usual hyperbolic rate equation with Hill coefficients close to unit. GSH saturation curves cannot be obtained at saturating CDNB because of the low solubility of CDNB. However, the cooperativity of all the Cys-47 mutants, measured in terms of n(H), does not change appreciably at fixed CDNB concentrations ranging between 0.1 and 1 mM. Therefore, under these conditions, an heterotropic effect by CDNB on the binding of GSH has not been observed. A possible homotropic behavior by CDNB has been also checked. Actually, an accurate estimate of the V(max) for CDNB required for the Hill calculation was not possible because of the substrate's low solubility. We obtained evidence of noncooperative behavior by CDNB only by examining a simple reciprocal plot 1/vversus 1/[CDNB] at fixed GSH concentration of 0.5 mM. No apparent deviation from linearity was observed for the C47S/C101S mutant (Fig. 2b) as well as for all other mutants (data not shown).


Figure 1: Homotropic behavior of C47S mutant. GSH saturation curves of C47S mutant and of wild type at 25 °C were obtained as described under ``Experimental Procedures.'' Initial velocities are expressed as DeltaA/min. GSH concentrations are expressed as mM. Each experimental point is the mean of five determinations. Error bars show the standard error of the mean. The data of C47S mutant (bullet) were fitted by nonlinear least squares regression analysis with . The solid line is the best fit. The dashed line represents the theoretical curve for a noncooperative GST with the same V(max) and [S](0.5) as the mutant. Data of wild type (up triangle) were fitted by nonlinear least squares regression analysis with assigning n(H) = 1 (rectangular hyperbola). The solid line is the best fit. Inset, Hill plots for C47S mutant (bullet) and for wild type (Delta). n(H) were 1.43 and 1.01, respectively.





Fig. 3), as well as for C47A and C47S/C101S mutants (data not shown) in the presence of hexylglutathione as inhibitor and GSH approximately at 0.1 [S](0.5). Maximal activations of 144% and 151% were found for C47A and C47S/C101S mutants, respectively. The wild type enzyme and the C101A mutant did not display any overreactivity (Fig. 3).


Figure 3: Activation by S-hexylglutathione. Experiments were performed at 25 °C as described under ``Experimental Procedures.'' The activity was assayed at fixed CDNB concentration (1 mM) and at about 0.1[S](0.5) GSH concentration. , wild type at 0.01 mM GSH; Delta, C47A/C101A mutant at 0.1 mM GSH; circle, C101A at 0.03 mM GSH; box, C47S mutant at 0.1 mM GSH.



GSH Binding

A few factors can simulate cooperativity. For example, a dimeric enzyme which has two independent subunits and two substrates, under certain conditions, may yield apparent positive cooperativity if it follows a steady-state random mechanism(27) . This event is improbable for GST P1-1 which obeys random rapid equilibrium kinetics(14) , but the possibility that the replacement of Cys-47 could induce a change of the kinetic mechanism cannot be absolutely ruled out. Furthermore, a substrate-induced change of a monomer-dimer equilibrium could be a second source of apparent cooperativity. Taking into account these possibilities we analyzed the isothermic binding of GSH on some mutants utilizing the quenching of intrinsic fluorescence due to the binding of this substrate(24) . The C47A and C47S mutants and the C47A/C101A double mutant exhibited sigmoidal isothermic binding curves. The Hill coefficients were 1.31, 1.36, and 1.51, respectively (Table 3), very close to that found with the kinetic procedure (see Table 2). Data referring to the double mutant are given in Fig. 4as a sigmoidal plot DeltaF(c)versus log[GSH]. These results are consistent with the idea that the binding of GSH on the first subunit induces a favorable conformational change on the second subunit which displays an increased affinity for GSH. Thus the possibility of an apparent kinetic cooperativity due to a steady-state random mechanism may be ruled out. Moreover, the Hill coefficients obtained by the fluorometric procedure (performed at about 0.05 mg/ml of GST) are similar to those obtained by the kinetic approach (performed at 1 µg/ml of GST), clearly indicating the absence of any monomer-dimer equilibrium in this phenomenon.





Figure 4: Cooperative binding of GSH on the C47A/C101A mutant. GSH binding was measured by fluorometry as described under ``Experimental Procedures''. GSH concentrations are expressed as µM. Data were fitted by nonlinear least squares regression analysis with . The solid line is the best fit. The dotted line and dashed line are the expected curves for n(H) = 1 (no cooperativity) and n(H) = 2 (infinite cooperativity), respectively.



Temperature Effect

On the assumption that the homotropic behavior found in the Cys-47 mutants is produced by a ligand-induced conformational change which is communicated to the nearby subunit, it is reasonable to expect that cooperativity slows down by enhancing the rigidity of the tertiary structure of the GST mutants. Steady-state kinetics of the GSH conjugation with CDNB by several Cys-47 mutants were studied in the temperature range of 10-35 °C. As reported in Fig. 5, both the C47S and, more evidently, C47A reduce their cooperative behavior at low temperatures. On the contrary the extent of the homotropic behavior of the double mutants remains quite high even at 10 °C. Apparently, lack of Cys-101 confers to the C47S and C47A mutants a higher flexibility that cannot be reduced by lowering the temperature near 10 °C. It results also that [S](0.5) values, which are an indication of the affinity for GSH, are greatly affected by temperature; all mutants displayed a similar temperature dependence of [S](0.5) and increased affinities for GSH at low temperatures. An interesting correlation can be made between these kinetic parameters. It appears that the cooperativity of mutants almost disappears when the affinity for GSH becomes similar to the wild type ([S](0.5) = 0.3-0.4 mM). This occurs at low temperatures only for single mutants of Cys-47 and never for double mutants. In other words a marked cooperativity is expressed only when [S](0.5) is quite higher (>0.5 mM) than that of the wild type (0.15 mM).

a



Figure 5: Temperature effect on cooperativity. Kinetic experiments were performed as reported under ``Experimental Procedures'' at the indicated temperatures. Data were fitted by linear least squares regression analysis with to calculate the Hill coefficients. [S](0.5) values for GSH were obtained from . Filled symbols indicate n(H) values; unfilled symbols indicate [S](0.5) values. a, data for C47S mutant (circles) and for C47S/C101S mutant (squares). b, data for C47A mutant (circles) and for C47A/C101A mutant (squares).




CONCLUDING REMARKS


Two microscopic models are commonly used to explain homotropic behavior in oligomeric enzymes. The first one assumes the enzyme to exist in two conformations, namely R and T, which are in equilibrium and differ in catalytic properties and (or) in substrate affinity (two-state model)(28) . Binding of the substrate shifts the T-R equilibrium toward the most active forms (R) and it originates nonhyperbolic kinetics. In the second model the enzyme may exist in intermediate conformations beside the T and R forms (sequential model) (29) . All the present data referring to the nonhyperbolic steady-state kinetics induced by replacement of Cys-47 in GST P1-1 can be well interpreted by the first model, although the sequential one is equally applicable. It appears that the substitution of Cys-47 yields a low-affinity conformation (T) which can be converted to a higher affinity conformation (R) (similar to that of wild type) upon binding with GSH, as appears in Fig. SI.


Scheme I:

No kinetic cooperativity has been observed in the wild type and in C101A and C101S mutants nor heterotropic and homotropic effects by CDNB in all mutants tested (see Fig. 2b). The independence of the observed cooperativity from CDNB binding or from some kinetic artifacts was also suggested by the cooperative isothermic binding of GSH as determined by fluorescence experiments (see Fig. 4and Table 3), as well as by the activation experiments (see Fig. 3). The higher values of Hill coefficients found for the double mutants compared with the single mutants, as well as the noncooperativity of the C101A and C101S mutants, point for a positive, but not crucial, contribution of the replacement of Cys-101 in the attainment of the R conformation upon GSH binding.

Is there some structural explanation for this induced cooperativity? As appears from crystallographic data of the enzyme in complex with s-hexylglutathione(11) , Cys-47 is placed close the G-site at 10.7 Å from the sulfur atom of the inhibitor at the end of the irregular helix alpha 2 (residues 35-44). The crystallographic temperature factors, experiments of limited proteolysis, and other spectroscopic data indicate that this region is highly mobile and it undergoes conformational changes upon binding of GSH(30, 31, 24) . The peculiar ion pair between Cys-47 and Lys-54 may act as an ``hinge'' which limits the number of potential conformations. Replacement of the sulfhydryl moiety of Cys-47 with the undissociated hydroxyl function of serine or its deletion, as occurs in the C47A mutant, suppresses this ionic bond. The resulting increased mobility is probably responsible both for the existence of a GSH low-affinity conformation and for a possible cooperative split to a high-affinity conformation upon GSH binding. With this model, the observed temperature dependence of the Hill coefficients (see Fig. 5) may be explained by assuming that at low temperatures the C47S and C47A mutants display lower mobility in this protein portion; the structure of the G-site and its affinity for GSH become similar to that of the wild type and the cooperativity disappears. If all the above conclusions are correct, we must find an induced cooperativity similar to that observed by replacement of Cys-47 when the counterpart in the ion pair with Cys-47, i.e. Lys-54 is substituted with a nonpositive residue. This demonstration will be given in the subsequent paper in this issue(22) .

Although it was well established the absence of any kinetic cooperativity for GST P1-1, the possibility of a structural communication between the two subunits has been previously suggested to occur in the horse erythrocyte isoenzyme which belongs to the class Pi. In two different papers we stressed the nonequivalence of its subunits in their reaction with sulfhydryl reagents(32, 33) ; possible explanations of this behavior included a nonsymmetrical association of identical subunits or a structural change of the unreacted subunit upon reaction of Cys-47 on the first subunit. In light of the present results, the last possibility appears reasonable.

Conversion of a noncooperative enzyme into a cooperative one by a mutation of a single amino acid residue is an interesting phenomenon; recently, it has been reported that mutation of a specific residue may introduce cooperativity into four previously noncooperative oligomeric enzymes. Some of these enzymes have been known to be evolutionary related to cooperative enzymes. This is the case of the trimeric aspartate transcarbamoylases (EC 2.1.3.2) from Bacillus subtilis and ornithine transcarbamoylase (EC 2.1.3.3) that become cooperative enzymes upon replacement of a single residue at the active site(34, 35) . On the other hand two dimeric enzymes, not related to allosteric enzymes, undergo induced cooperativity; glutathione reductase (EC 1.6.4.2) displays homotropic behavior when a glycine residue, located in the dimer interface, is replaced with a tryptophan residue(36) ; tyrosyl-tRNA synthetase (EC 6.1.1.1.) shows cooperativity upon mutation of Lys-233, a residue involved in the substrate binding (37) .

GST is not related to allosteric proteins. Moreover the mutated residue that triggers cooperativity (Cys-47) is not located in the subunit interface and is not inside the active site. These peculiarities make GST a new and interesting model for induced cooperativity.

From a more general point of view the above findings support the attractive hypothesis that some oligomeric proteins may have the potential to be allosterically controlled. This potential may be expressed by a evolutionary mutation of one or a few amino acids which can transform the oligomeric enzyme into a regulatory enzyme. This did not occur for GSTs; there is no apparent utility of allosteric modulation of activity for an enzyme whose role is to detoxify potentially dangerous chemicals.


FOOTNOTES


*
This work was partially supported by a grant from Consiglio Nazionale delle Ricerche, Progetto Finalizzato: ``Applicazioni Cliniche della Ricerca Oncologica.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biology, University of Rome, ``Tor Vergata,'' Viale della Ricerca Scientifica, 00133-Rome Italy. Tel.: 06-72594375; Fax: 06-2025450.

Wellcome Australian Senior Research Fellow and supported by the Anti-cancer Council of Victoria.

(^1)
The abbreviations used are: GST, glutathione transferase; CDNB, 1-chloro-2,4-dinitrobenzene. Mutated enzymes are designated using one-letter code for the original amino acid (before the residue number) and for the replaced amino acid after the residue number.


ACKNOWLEDGEMENTS


We thank Prof. Paolo Ascenzi for helpful discussion of the manuscript.


REFERENCES


  1. Jakoby, W. B. (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 46, 383-414 [Medline] [Order article via Infotrieve]
  2. Jakoby, W. B., Ketterer, B., and Mannervik, B. (1984) Biochem. Pharmacol. 33, 2539-2540 [Medline] [Order article via Infotrieve]
  3. Mannervik, B. (1985) Adv. Enzymol. Relat. Areas Mol. Biol. 57, 357-417 [Medline] [Order article via Infotrieve]
  4. Mannervik, B., and Danielson, U. H. (1988) CRC Crit. Rev. Biochem. 23, 283-337 [Medline] [Order article via Infotrieve]
  5. Pickett, C. B., and Lu, A. Y. H. (1989) Annu. Rev. Biochem. 58, 743-764 [CrossRef][Medline] [Order article via Infotrieve]
  6. Armstrong, R. N. (1991) Chem. Res. Toxicol. 4, 131-140 [Medline] [Order article via Infotrieve]
  7. Mannervik, B., Alin, P., Guthenberg, C., Jensson, H., Tahir, M. K., Warholm, M., and Jornvall, H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7202-7206 [Abstract]
  8. Meyer, D. J., Coles, B., Pemble, S. E., Gilmore, K. S., Fraser, G. M., and Ketterer, B. (1991) Biochem. J. 274, 409-414 [Medline] [Order article via Infotrieve]
  9. Sinning, I., Kleywegt, G. J., Cowan, S. W., Reinemer, P., Dirr, H. W., Huber, R., Gilliland, G. L., Armstrong, R. N., Ji, X., Board, P. G., Olin, B., Mannervik, B., and Jones, T. A. (1993) J. Mol. Biol. 232, 192-212 [CrossRef][Medline] [Order article via Infotrieve]
  10. Reinemer, P., Dirr, H. W., Ladenstein, R., Shaffer, J., Gallay, O., and Huber, R. (1991) EMBO J. 10, 1997-2005 [Abstract]
  11. Reinemer, P., Dirr, H. W., Ladenstein, R., Huber, R., Lo Bello, M., Federici, G., and Parker, M. W. (1992) J. Mol. Biol. 227, 214-226 [Medline] [Order article via Infotrieve]
  12. Ji, X., Zhang, P., Armstrong, R. N., and Gilliland, G. L. (1992) Biochemistry 31, 10169-10184 [Medline] [Order article via Infotrieve]
  13. Danielson, U. H., and Mannervik, B. (1985) Biochem. J. 231, 263-267 [Medline] [Order article via Infotrieve]
  14. Ivanetich, K. M., and Goold, R. D. (1989) Biochim. Biophys. Acta 998, 7-13 [Medline] [Order article via Infotrieve]
  15. Schramm, V. L., McCluskey, R., Emig, F. A., and Litwack, G. (1984) J. Biol. Chem. 259, 714-722 [Abstract/Free Full Text]
  16. Ivanetich, K. M., Goold, R. D., and Sikakana, C. N. T. (1990) Biochem. Pharmacol. 39, 1999-2004 [CrossRef][Medline] [Order article via Infotrieve]
  17. Jakobson, I., Warholm, M., and Mannervik, B. (1979) J. Biol. Chem. 254, 7085-7089 [Abstract]
  18. Tamai, K., Shen, H., Tsuchida, S., Hatayama, I., Satoh, K., Yasui, A., Oikawa, A., and Sato, K. (1991) Biochem. Biophys. Res. Commun. 179, 790-797 [Medline] [Order article via Infotrieve]
  19. Kong, K. H., Inoue, H., and Takahashi, K. (1991) Biochem. Biophys. Res. Commun. 181, 748-755 [Medline] [Order article via Infotrieve]
  20. Ricci, G., Del Boccio, G., Pennelli, A., Lo Bello, M., Petruzzelli, R., Caccuri, A. M., Barra, D., and Federici, G. (1991) J. Biol. Chem. 266, 21409-21415 [Abstract/Free Full Text]
  21. Lo Bello, M., Parker, M. W., Desideri, A., Polticelli, F., Falconi, M., Del Boccio, G., Pennelli, A., Federici, G., and Ricci, G. (1993) J. Biol. Chem. 268, 19033-19038 [Abstract/Free Full Text]
  22. Lo Bello, M., Battistoni, A., Mazzetti, P., Board, P. G., Muramatsu, M., Federici, G., and Ricci, G. (1995) J. Biol. Chem. 270, 1249-1253 [Abstract/Free Full Text]
  23. Habig, W. H., and Jakoby, W. B. (1981) Methods Enzymol. 77, 398-405 [Medline] [Order article via Infotrieve]
  24. Caccuri, A. M., Aceto, A., Rosato, N., Di Ilio, C., Piemonte, F., and Federici, G. (1991) Ital. J. Biochem. 40, 304-311 [Medline] [Order article via Infotrieve]
  25. Collins, K. D., and Srark, G. R. (1971) J. Biol. Chem. 246, 6599-6605 [Abstract/Free Full Text]
  26. Stebbins, J. W., Xu, W., and Kantrowitz, E. R. (1989) Biochemistry 28, 2592-2600 [Medline] [Order article via Infotrieve]
  27. Segel, I. H. (1975) Enzyme Kinetics , pp. 460-461, John Wiley & Sons, New York
  28. Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118 [Medline] [Order article via Infotrieve]
  29. Koshland, D. E., Jr., Nemethy, G., and Filmer, D. (1966) Biochemistry 5, 365-385 [Medline] [Order article via Infotrieve]
  30. Lo Bello, M., Pastore, A., Petruzzelli, R., Parker, M. W., Wilce, M. C. J., Federici, G., and Ricci, G. (1993) Biochem. Biophys. Res. Commun. 194, 804-810 [CrossRef][Medline] [Order article via Infotrieve]
  31. Vander Jagt, D. L., Wilson, S. P., and Heidrich, J. E. (1981) FEBS Lett. 136, 319-321 [CrossRef][Medline] [Order article via Infotrieve]
  32. Ricci, G., Del Boccio, G., Pennelli, A., Aceto, A., Whitehead, E. P., and Federici, G. (1989) J. Biol. Chem. 264, 5462-5467 [Abstract/Free Full Text]
  33. Del Boccio, G., Pennelli, A., Whitehead, E. P., Lo Bello, M., Petruzzelli, R., Federici, G., and Ricci, G. (1991) J. Biol. Chem. 266, 13777-13782 [Abstract/Free Full Text]
  34. Stebbins, J. W., and Kantrowitz, E. R. (1992) Biochemistry 31, 2328-2332 [Medline] [Order article via Infotrieve]
  35. Kuo, L. C., Zambidis, I., and Caron, C. (1989) Science 245, 522-524 [Medline] [Order article via Infotrieve]
  36. Scrutton, N. S., Deonarain, M. P., Berry, A., and Perham, R. N. (1992) Science 258, 1140-1143 [Medline] [Order article via Infotrieve]
  37. First, E. A., and Fersht, A. R. (1993) Biochemistry 32, 13651-13657 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.