site-directed mutagenesis of glutathione s-transferase yaya · site-directed mutagenesis of...

THE J O U R N A L 0 1992 by The American Society for Biochemistry and Molecular Biology,

OF BIOLOGICAL CHEMISTRY Inc.

Vol. 267, No. 28, Issue of October 5, pp. 19866-19871,1992 Printed in U. S. A .

Site-directed Mutagenesis of Glutathione S-Transferase YaYa IMPORTANT ROLES OF TYROSINE 9 AND ASPARTIC ACID 101 IN CATALYSIS*

(Received for publication, May 22, 1992)

Regina W. Wang$#, Deborah J. Newton$, Su-Er Wu €€uskey$, Brian M. McKeeverll, Cecil B. Pickettll, and Anthony Y. H. Lu$ From the $Department of Animal & Exploratory Drug Metabolism, TIfepartment of Siophysical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900 and the IlMerck Frosst Center for Therapeutic Research, Pointe-Claire-Dorval, Quebec H9R4P8, Canada

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Re- placement of tyrosine 9 with phenylalanine (YgF), threonine (YgT), histidine (YgH), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK, values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK, value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pK, of the sulfhydryl group of glutathione, presum- ably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine.

To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, DlOlE, and DlOlN) indicate that only DlOlN exhibited a &fold increase in K , toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays a n important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.

Glutathione S-transferases (EC 2.5.1.18) are a family of enzymes that catalyze the nucleophilic addition of glutathione to electrophilic substrates (1-7). In mammalian tissues, the cytosolic GSTs’ are classified into three distinct gene classes

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be ad- dressed RY80A12, Dept. of Animal & Exploratory Drug Metabolism, Merck Research Laboratories, P.O. Box 2000, Rahway, N J

The abbreviations used are: GST, glutathione S-transferase; CDNB, l-chloro-2,4-dinitrobenzene; PCR, polymerase chain reaction; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

07065-0900.

(a , p, and E ) based on their substrate specificity, immunochemical reactivity and other properties, and their primary amino acid sequences (4). The structure and function rela- tionships of various GSTs have been investigated by chemical modification (8-17), GSH analogs (18-20), affinity labeling (21), and site-directed mutagenesis (22-31). More recently, the three-dimensional structure of the ?r class GST from pig lung complexed with glutathione sulfonate has been reported (32).

Despite the fact that all GSTs require GSH for catalytic activity, no consensus sequence for the GSH binding site has been noted from the amino acid sequences of all known GSTs. Indeed, only about 5% of the amino acids are conserved in all the GSTs (4). These conserved amino acids have been the target of site-directed mutagenesis for defining the site of GSH binding and determining their role in catalysis.

In our laboratory, we have focused on the structure and function relationship of the a class rat liver GST YaYa.* In earlier studies, we demonstrated that histidine (at positions 8, 143, and 159), cysteine (at positions 18 and 112), and tryptophan (at position 21) are not involved in catalysis of GST YaYa (22, 31). In this study, we investigated the roles of tyrosine 9 and several conserved aspartic acids in YaYa- mediated reactions. Our results indicate that tyrosine 9 plays an important role in catalysis, whereas aspartic acid 101 is involved in GSH interaction.

EXPERIMENTAL PROCEDURES

Materials

Reagents used in this study were obtained from the following sources: restriction endonuclease SalI, EcoRI, and T4 DNA ligase from Bethesda Research Laboratories; Gene Amp kits from Perkin- Elmer Cetus Instruments; DNA sequencing kits from United States Biochemical Corp.; GSH, CDNB, NADPH, S-hexylglutathione, S- hexylglutathione-epoxyagarose, and hematin from Sigma; A5-androstene-3,17-dione from Steraloids, Inc.; GSH reductase from Boehrin- ger Mannheim; cumene hydroperoxide from Aldrich; deoxyadenosine 5’-~~-[~~S]thiotriphosphate and lZ5I-labeled protein A from Amersham; Escherichia coli strain AB1899 (the ion” protease-deficient strain) from Genetic Stock Center, Yale University (New Haven, CT).

Methods Oligonucleotide Primer Design and PCR Mutagenesis-oligonucle-

otide primers for PCR mutagenesis were synthesized on a Cyclone DNA synthesizer (Biosearch, Inc.). Primer A (5”ACACCGAATTC- ATGTCTGGGAAGCCAGTGCT-3’) and primer B (S’AGGTCGT- CGACCTAAAACTTGAAAACCTTCCTTG-5’) are the 5’-end forward and 3’-end inverse sequences of Ya cDNA with added EcoRI and SalI restriction sites, respectively. Site-directed mutagenesis was

* Subunit Ya is equivalent to subunit 1, the proposed nomenclature (33).

19866

Roles of Tyrosine and Aspartic Acid in Catalysis of GST YaYa 19867

performed as described previously (22). Point mutations in cDNA were generated by PCR using forward and inverse mutagenic primer pairs containing the mismatched codons on the template with wild type Ya cDNA (34). The mutated cDNA was inserted into expression vector pKK 2.7 (34,35). The constructed expression plasmids bearing the desired codon changes were used to transform E. coli strain AB 1899. Positive colonies were selected by ampicillin resistance and PCR screening procedures as described (22). The entire cDNA en- coding GST YaYa mutants were sequenced (22, 36) in order to confirm the desired base substitutions and to ensure that no other mutations were introduced into the nucleotide sequence during the PCR.

Purification ofthe Expressed Enzyme-Cells from 1-liter overnight cultures were harvested by centrifugation at 10,000 X g for 20 min and disrupted by sonication in 10 mM sodium phosphate buffer, pH 7.4, containing 5 mM EDTA. The cell debris and unbroken cells were removed by centrifuging a t 10,000 X g for 25 min, and the resulting supernatant fractions were centrifuged a t 100,000 X g for 60 min to obtain the cytosolic fraction. The cytosolic fractions were concen- trated with an Amicon PM-10 membrane and subjected to a Sephadex G-50 column. The fractions with transferase activities were pooled and loaded onto a S-hexylglutathione-epoxy-agarose column. The expressed proteins, which were bound to the affinity column, were eluted with 2.5 mM GSH and 5 mM S-hexylglutathione in a 25 mM Tris-HCI buffer, pH 8.0, containing 0.2 M KC1 (34, 37).

Immunoblot Analysis-Cytosolic fractions from each culture were subjected to 12% SDS-polyacrylamide gel electrophoresis according to Laemmli (38) and transferred to a nitrocellulose membrane (Schleicher and Schuell) using the method of Towbin et a/. (39). The polyclonal antibodies against the Ya subunit (40) were used to detect the expressed proteins in E. coli cytosolic fractions.

Assays of Enzyme Actiuity-GST conjugating activities, peroxidase activities, and isomerase activities, with CDNB, cumene hydroperoxide, and A"-androstene-3,17-dione as the respective substrates, were determined as described (41-44). Kinetic constants for CDNB and GSH were measured with 2 mM GSH or 2 mM CDNB and varying concentrations of the other substrate ranging from 0.1-2 mM. Kinetic parameters were obtained from hyperbolic saturation curves by least- squares fit of the data to the Michaelis-Menten equation. The pH rate profiles for the conjugating reactions of GSH with CDNB catalyzed by purified wild type and mutant enzymes were conducted in 0.25 M bis-tris propane buffer using the substrate concentrations shown in the figures. The rates of all the enzymatic reactions in the study were obtained by subtracting the nonenzymatic reaction rates from the observed reaction rates. Protein concentrations were determined by the method of Lowry et al. (45) with bovine serum albumin as a standard.

Fluorescence Quenching Titration-The hematin solution was prepared as described (46, 47), and the concentration was determined from the absorbance (at 400 nm) of a dilution of the stock solution in 40% dimethyl sulfoxide ( e = 180 mM" cm") (48). Fractional quenching of the intrinsic protein fluorescence upon addition of hematin was measured a t excitation wavelength 282 nm and emission wavelength 330 nm. The hematin concentration which caused 50% fluorescence quenching of the enzymes was determined from hyperbolic saturation curves which were constructed by plotting the fractional quenching versus the concentration of hematin added to the protein sample.

RESULTS

Expression of Mutant Enzymes in E. coli-To establish the role of tyrosine 9 in catalysis, Y9F and Y9T mutants were generated to determine the importance of the hydroxyl group. Histidine, which can function as a general base, and valine, a neutral amino acid, were also used to replace tyrosine 9. For the role of aspartic acid in GSH binding, mutants were designed to retain the negative charge (glutamic acid) or to eliminate the charge (asparagine) on the side chain. When the cytosolic fractions containing either the wild type enzyme or the mutant enzymes were examined by immunoblot analysis, the mutant proteins co-migrated with the wild type protein with comparable levels of expression (Fig. 1). The CDNB- conjugating activities of t.he wild type and mutant enzymes were also examined in the cytosolic fractions (Table I). The replacement of tyrosine 9 with phenylalanine, threonine, his-

FIG. 1. Immunochemical analysis of the cytosolic fraction of wild type and mutant enzymes. The cytosolic fractions of individual cultures harboring the wild type and mutant enzymes were immunoblotted with polyclonal antibodies against YaYa.

TABLE I Tvrosine and aswartic acid mutants

~

Mutant Amino acid Codon CDNB-conjugating activity changed changed of cytosolic fraction

Y9F Tyr-9 + Phe Y9T Tyr-9 + Thr Y9H Tyr-9 + His Y9V Tyr-9 + Val

D93N Asp-93 + Asn

DlOlN Asp-101 + Asn

D61N Asp-61 + Asn

D157N ASD-157 + Asn

D93E Asp-93 + GIU

DlOlE Asp-101 + Glu

D61E ASP-61 + GIU

D157E ASP-157 + GIU

TAC + TTC TAC + ACT TAC + CAT TAC + GTT GAC + GAA GAC + AAC GAT .-, GAA GAT + AAC GAC + GAA GAC + AAC GAC + GAA GAC + AAC

% uild type" 2 2 2

89 0.1

35 87 25 87 58 44 0.1

Activity for the cytosolic fraction of wild t-ype enzyme was 0.18 ymol/min/mg of protein.

tidine, or valine resulted in dramatic decreases in enzyme activities. These results demonstrate the specific requirement of the hydroxyl group of tyrosine 9 in the catalytic mechanism of rat GST YaYa. When aspartic acid was replaced with glutamic acid, the mutants maintained most of the enzyme activities. However, much lower activities were noted with the asparagine mutants.

To further characterize the enzymes, two of the tyrosine mutants (Y9F and Y9T) and four of the aspartic acid mutants (D93E, D93N, DlOlE, and DlO1N) were purified to apparent homogeneity by affinity chromatography. In spite of the apparent loss of activity for D157N, aspartic acid 157 mutants were not pursued at this time since this amino acid does not appear to be positioned at the GST active site in the proposed three-dimensional structure (32). The CDNB-conjugating activities, isomerase activities, and peroxidase activities of the purified enzymes were determined. As shown in Table 11, all purified tyrosine mutants have very low catalytic activities for all three enzymatic reactions. Purified aspartic acid mutants, on the other hand, exhibited partial enzymatic activities.

Characterization of Tyrosine Mutants-The kinetic parameters of Y9F and Y9T mutants for the conjugation reaction between CDNB and GSH were determined in 0.1 M potassium phosphate buffer, pH 6.5, by standard assay conditions. The catalytic efficiencies (kcat/&) toward CDNB and GSH for these mutants were much lower than the wild type GST as shown in Table 111. For mutant Y9F, the K, values toward CDNB and GSH were comparable to those obtained for the

19868 Roles of Tyrosine and Aspartic Acid i n Catalysis of GST Y a Y a

wild type enzyme. Although an increase in K, for CDNB was noted for Y9T, a very large standard deviation was also observed (due to extremely low activity); therefore, it is dif- ficult to assess the significance of this increase. In any event, i t is reasonable to state that poor catalytic efficiencies for Y9F and Y9T are largely due to a marked decrease in kc,,.

In order to examine the mechanism of the involvement of tyrosine 9 in catalysis, pH rate profiles were measured as described by Huskey et al. (49). The pH-dependent rates for the enzymes were measured under saturating conditions of one substrate (either CDNB or GSH) and less than saturating levels of the other substrate. For chemical reactions, least- squares fit of the data to Equation 1 was used to obtain the estimated K, value in a simple one-pK, model. For enzymatic reactions, the pH rate data can be expressed in terms of the two-pKa model of Equation 2. Only the pKa2 values in the

uo = [ G S H l o [ C D N B l o ~ ~ K ~ / ~ K ~ + [H']) (1)

vo = ~[H'lKo,/(Ko,Ka, + (Ka2 + KO?) [H'l + [H+12) (2)

enzymatic reaction are relevant to the present study since earlier pH studies (29, 49, 50) have shown that a pK, of 7 reflects the ionization of GSH in the enzyme-substrate complex. The significance of pKal (approaching 10) in the conjugation reaction is unclear.

The nonenzymatic reaction between GSH and CDNB reached maximum activity around pH 9 to 10 with estimated pK, values of 9 (Fig. 2, B and D). In contrast, the YaYa catalyzed reaction (Fig. 2, A and C, and Table IV) exhibited a pH optimum of 8.5 with pK, values of approximately 7, a shift of 2 units as previously reported (49, 50). Interestingly, when Y9F and Y9T mutants were used to catalyze the conjugation reaction (Fig. 2, A and C, and Table IV), the pH optimum was shifted back to around 9 to 10 with estimated pK, values of 8.5 to 8.6 for Y9F and 8.6 to 8.7 for Y9T. These data are similar to those found for the chemical reaction (estimated pK, values, 8.9 to 9.1). Thus, it is clear that tyrosine 9 is directly responsible for the shift of the pK, of the sulfhydryl group of GSH from approximately 9 in the chemical reaction to approximately 7 in the YaYa-catalyzed

TABLE I1 Enzyme activit-y of purified wild type and mutant erzz.ymes

Enzyme CDNB-conjugating Isomerase Peroxidase activitv activitv activitv

% wild type"

Tyrosine mutant Y9F 5 5 3 Y 9 T 4 4 2

D93E 75 57 66 D93N 42 52 33 DlOlE 60 46 51 DlOlN 21 13 28 Conjugating, isomerase, and peroxidase activities of purified wild

type GST were 26.3, 1.22, and 2.66 wmol/min/mg of protein, respec-

Aspartic acid mutant

tively.

reaction. I t should be noted that, in these experiments, higher concentrations of mutant enzymes were used in comparison to the wild type enzyme and that nonenzyme reaction rates were subtracted from the observed rates to obtain the enzymatic rates shown in the figures.

Characterization of Aspartic Acid Mutants-The kinetic parameters of aspartic acid mutants for the conjugation reaction between CDNB and GSH were obtained for the purified enzymes (Table V). Aspartic acid 93 does not appear to play any significant role in catalysis since no significant changes in K,,, or kcat/K,,, were noted for D93E and D93N. As for aspartic acid 101, replacement by glutamic acid (DlOlE) did not cause any significant alterations in substrate binding nor catalytic efficiency. In contrast, a 5-fold increase in K, for GSH and a very significant decrease in k,,,/K, were noted for mutant DlOlN. In addition to this increase in K, toward GSH, poor binding of this mutant enzyme to the S-hexylglutathione affinity column was noted during purification. Un- like the wild type and DlOlE, more than 70% of DlOlN activity was not retained by the affinity column and what was retained was easily washed out from the column (Table VI). Thus, the negative charge of aspartic acid 101 appears to be involved in the binding of GSH to the active site of GST YaYa.

To examine the possibility that aspartic acid 101 may also be involved in the lowering of the pK, of the sulfhydryl group of enzyme-bound GSH, pH rate profiles were conducted. The pK, values estimated for the wild type, DlOlE, and DlOlN were very similar (Table IV); no major shift in the pK, was noted (Fig. 3).

Binding of Hematin-The possible involvement of tyrosine 9 and aspartic acid 93 and 101 in heme binding was studied by fluorescence quenching to determine the quantitative binding of hematin to the wild type and mutant enzymes. The heme concentration required to give 50% fluorescence quenching was determined and is presented in Table VII. There was no significant changes in fluorescence quenching, indicating that tyrosine 9 and aspartic acid 93 and 101 are not involved in heme binding.

DISCUSSION

Mechanistic studies by Armstrong and co-workers (20, 50) suggest that the thiolate anion is the predominant species of enzyme-bound GSH at physiological pH. Thus, an important feature of the mechanism in the GST-catalyzed reaction is the apparent stabilization of the thiolate anion and the lowering of the pK, of the sulfhydryl group of GSH from 9 in aqueous solution to approximately 7 of the enzyme-bound form (3, 20, 50). In addition, GSTs may also enhance the reactivity of the thiolate by shielding it from solvent (3, 49). Kinetic solvent isotope effect studies estimated that in the chemical reaction, the extent of the desolvation of the thiolate anion in the transition state is about 34%, whereas in the enzyme-catalyzed reaction, the desolvation is almost complete (49). These results imply that the amino acid residue(s) in

TABLE I11 Kinetic constants of purified enzymes /or wild type and tyrosine mutants

Kinetic constants for CDNB and GSH were obtained as described under "Experimental Procedures." CDNB GSH

Enzyme K", kcat kcatlKm Km kcat kes/Km

m M S" mM" s" m M S" m M " s "

Wild type 0.56 f 0.06 52 f 2 92 k 6 0.31 f 0.03 51 f 1.5 164 f 12 Y9F 0.23 f 0.04 1.1 * 0.1 4.8 k 0.6 0.19 f 0.07 0.8 f 0.1 4.5 f 1.1 Y9T 2.32 f 1.39 2.6 f 1.2 1.1 k 0.2 0.28 * 0.10 1.0 f 0.1 3.7 f 0.9

Roles of Tyrosine and Aspartic Acid i n Catalysis of GST YaYa 19869

/

I 9

I [GSH] = 2 mM

10 11

PH

1 ' 1 ' 1 ' 1

C 0 [CDNE] = 2 mM IGSHI = 0.2 mM . . 1

7 8 9 PH

10 11 12

l[CONE] = 2 rnM [GSH] = 0.2 mM

7 S 9 10 11

TABLE IV Kinetic parameters for the reaction of GSH and CDNB catalyzed by

GST wild type and mutant enzymes Parameters K,,, and KO2 were obtained from least-squares fits of

the data in Figs. 2, A and C, and 3, A and B , to Equation 2. Error limits are estimates of standard deviations.

Enzyme [CDNB] [GSH] PK., PKO,

m M m M

Wild type 0.1 2 6.8 k 0.2 9.9 + 0.2 Y9F 0.1 2 8.6 f 0.2 10.8 k 0.3 Y9T 0.1 2 8.6 f 0.5 9.7 f 0.5 Wild type 2 Y9F

0.2 6.7 ? 0.3 9.9 f 0.2 2 0.2 8.5 f 0.5 10.9 f 0.7

Y9T 2 0.2 8.7 + 0.4 10.2 f 0.4 Wild type 0.06 1 DlOlE 0.06 1

7.2 -t 0.3 9.1 ? 0.3

DlOlN 0.06 1 7.0 ? 0.1 9.5 ? 0.1 7.5 f 0.1 9.3 f 0.2

Wild type 1.5 0.1 7.2 k 0.2 9.1 k 0.1 DlOlE 1.5 DlOlN

0.1 7.1 f 0.2 9.1 f 0.2 1.5 0.1 7.7 ? 0.2 9.2 ? 0.2

the active site of GSTs may serve to stabilize the thiolate anion. However, these studies do not address specifically the chemical nature of the amino acid residues involved in stabilization.

Recently, Reinemer et al. (32) presented a three-dimensional structure of the 7r class GST from pig lung complexed with glutathione sulfonate. They reported that the sulfonate group of the molecule is located within hydrogen bonding contact of the hydroxyl group of an active site tyrosine (at position 7). This information has prompted site-directed mutagenesis studies of tyrosines located at the active site of human a GST Al-1 (27), human 7r GST (30), and rat p class GST 3-3 (29), residue positions 8, 7, and 6, respectively. Replacement of all these tyrosines by phenylalanine resulted in a marked decrease in their catalytic activities. In our study with rat a class GST YaYa, tyrosine 9 was replaced by phenylalanine, threonine, histidine, and valine. All four mutants exhibited less than 5% of the wild type catalytic activity. Kinetic studies using purified Y9F indicate that the K, values toward CDNB and GSH for this mutant enzyme are relatively the same as the wild type enzyme. Thus, the drastic change in its catalytic efficiency is primarily due to the decrease in kc,,, consistent with the results obtained with the tyrosine mutants of other GSTs (27, 29, 30). These results clearly indicate that tyrosine 9 of rat a class GST YaYa plays an important role in catalysis.

The pH rate profile studies showed that the pK, of the GSH thiol bound to the wild type enzyme was about 7. In contrast, the pK, values of the GSH thiol bound to Y9F and Y9T mutants were 8.5 to 8.7, similar to the pK, of the thiol (8.9 to 9.1) in the chemical reaction. Therefore, tyrosine 9 is clearly responsible for the lowering of the pK, of the sulfhydryl group of GSH from 9 in the chemical reaction to 7 in the enzyme-catalyzed reaction. By replacing tyrosine 9 with phenylalanine or threonine, the mutant enzymes achieve only a slight rate enhancement but retain the characteristics of the chemical reaction. Whether tyrosine 9 is also responsible for

FIG. 2. pH rate profiles for the reaction of GSH with CDNB catalyzed by wild type, Y9F, and Y9T enzymes. Each 1-ml reaction contained 5.4 pg of wild type enzyme (O"-O), 16 pg of Y9F (0-"o), or 16 pg of Y9T (O---O) in 0.25 M bis-tris propane using the substrate concentrations of 0.1 mM CDNB and 2 mM GSH ( A ) or 2 mM CDNB and 0.2 mM GSH (C). Nonenzymatic reaction rates for both sets of substrate concentrations, shown in panels B and D, have been subtracted from observed rates to obtain the enzymatic rates for panels A and C. Note: higher concentrations of mutant enzymes than that of wild type enzyme were used in the assays.

19870 Roles of Tyrosine and Aspartic Acid in Catalysis of GST YaYa

TABLE V Kinetic constants of purified enzymes for wild type and aspartic acid mutants

Kinetic constants for CDNB and GSH were obtained as described under "Experimental Procedures."

Enzyme CDNB GSH

K , k., k m f K m K , kcat kcdKrn m M S" mM" s-l m M S" mM"s"

Wild type 0.56 ? 0.06 52 ? 2 92 f 6 0.31 f 0.03 51 f 2 164 f 12 D93E 0.54 k 0.07 31 f 2 58 f 5 0.33 f 0.08 49 f 3 D93N

152 f 25 0.60 f 0.16 42 f 4 70 f 12 0.42 ? 0.18 35 ? 5

DlOlE 83 rf: 25

0.32 f 0.08 28 ? 2 85 f 15 0.40 f 0.10 45 f 4 113 rf: 19 DlOlN 0.60 ? 0.09 19 f 1 31 f 3 1.49 k 0.38 25 k 4 16 rf: 2

TABLE VI The comparison of activity ratio in flow-through fractions to eluted

fractions for wild type and aspartic acid 101 mutants Activity ratio of

to GSH-eluted fraction flow-through fraction Enzyme

Wild type DlOlE DlOlN

0.24 0.36 2.50

'6 u 7

a 9 10

PH

0.75 1 . 1 '

B [GSH] = 0 1 mM [CDNB] = 1.5 rnM

0.60 - 0

(I)

5 0.45 . - .- 21 0 - - f 0.30 - m c .- - ._ -

0.15

Of3 7 8 9 10

PH

FIG. 3. pH rate profiles for the reaction of GSH with CDNB catalyzed by wild type, DlOlE, and DlOlN enzymes. A, each 1-ml reaction contained 3 pg of wild type enzyme (0--O), 3.5 pg of DlOlE (0"-O), or 14.7 pg of DlOlN (0--0) in 0.25 M bis-tris propane using 0.06 mM CDNB and 1 mM GSH as substrates. B, each 1-ml reaction contained 1.5 pg of wild type enzyme (0--O), 3.5 pg of DlOlE (0-"o), or 10.5 pg of DlOlN (0--0) in 0.25 M bis-tris propane using 1.5 mM CDNB and 0.1 mM GSH as substrates. Note: higher concentrations of mutant enzymes than that of wild type enzyme were used in the assays, and the nonenzymatic reaction rates have been subtracted from the enzymatic rates.

TABLE VI1 Binding of hematin to GST wild type and mutant enzymes

Hematin concentrations used, 0-2 p ~ ; wild type or mutated GST, 0.3 pM.

Enzyme Heme concentration to give 50% fluorescence quenching

P M

Wild type 0.24 f 0.04 Tyrosine mutant

0.24 f 0.03 0.15 f 0.01

0.19 f 0.04 0.20 f 0.02 0.17 f 0.02 0.16 ? 0.02

Y9F Y9T

D93E D93N DlOlE DlOlN

Aspartic acid mutant

the enhanced thiolate desolvation by GST remains to be determined.

Several mechanisms have been proposed to explain the involvement of the active site tyrosine in the lowering of the pK, of the thiol group of bound GSH (29, 32). One possibility involves hydrogen bonding between tyrosine 9 and the sulfur of GSH to stabilize the thiolate anion. Using the information derived from the x-ray crystallography for the GST 3-3 binary complex (29), our proposed model was constructed so that the hydroxyl group of tyrosine 9 in YaYa is within hydrogen bonding distance to the bound GSH thiol (Fig. 4A). Hydrogen was only included as a visual aid to position tyrosine 9 with respect to the substrate. In contrast, the mutations either do not have a hydrogen bonding functional group (Y9F, Fig. 4B), or, when modeled on an active site tyrosine-GSH template, they are simply too far from the GSH thiol to provide any stabilizing influence (Y9H, Fig. 4C; and Y9T, Fig. 40). The other possibility suggests that tyrosine 9 may serve as a general base to facilitate the removal of the proton of GSH. Although the pKn of tyrosine is too high for tyrosine to serve such a function, this mechanism cannot be totally ruled out since the pK, of tyrosine 9 in the active site is still unknown. Further studies must be carried out to determine whether deprotonated tyrosine exists in the active site of GST YaYa a t physiological pH.

Ionic interaction may play an important role in the binding of GSH to GST (18, 19). As reported by Stenberg et al. (28), the conserved arginine residues at positions 13, 20, and 60 in human a GST t may contribute to the binding affinity of GSH. In our study, aspartic acid 101 appears to play an important role in GSH interaction to rat GST YaYa. This conclusion is based upon the observation that replacement of aspartic acid at position 101 with glutamic acid resulted in no significant change in catalytic efficiency or in the K,,, values for CDNB and GSH, whereas replacement by asparagine caused a significant decrease in kcat. Most importantly, the K,,, for GSH was increased approximately &fold for DlOlN.

Roles of Tyrosine and Aspartic Acid in Catalysis of GST YaYa 19871

A /!"

C /!"

I

w s n

B k

I '. .. ,..

FIG. 4. Schematic models depicting the interaction between tyrosine 9 (or its replacement) of GST YaYa with GSH. A, approximate position of tyrosine 9 with respect to the GSH substrate in the active site. The distance between the hydroxyl oxygen and the thiol sulfur atoms is fixed at 3.2 A based on the structure of the GST 3-3 complex (29). B, replacing tyrosine with phenylalanine at position 9 removes the hydroxyl group but does not allow enough room for a water molecule to occupy the same position. C , replacing tyrosine with histidine a t position 9 introduced a much stronger base that still requires major conformational adjustments to bring the imidazole group close enough to the GSH thiol. D, replacing tyrosine with threonine a t position 9 requires major conformational adjustments so that the hydroxyl group can act as a hydrogen bond donor to stabilize the thiolate anion. Dot-surface figures were prepared on a Silicon Graphics IRIS-4 work station using the QUANTA software package from Polygen Corp.

Elimination of the negative charge also caused decreased binding of the mutant enzyme to the GSH affinity column. No significant pK, shift of the enzyme-bound GSH thiol was noted for DlOlN and DlOlE, indicating that aspartic acid 101 is not involved in the lowering of the pK, of the sulfhydryl group of enzyme-bound GSH. Since the three-dimensional structure of rat GST YaYa is still under investigation, direct confirmation of an interaction between aspartic acid 101 and GSH remains to be established. Furthermore, additional experiments are needed to define the role of Asp-157 in catalysis.

Acknowledgments-We thank Dr. John Hayes for supplying antibodies and Terry Rafferty for assistance in the preparation of this manuscript.

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