Site-directed mutagenesis of glutathione S-transferase YaYa: Functional studies of histidine, cysteine, and tryptophan mutants

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 297, No. 1, August 15, pp. 86-91, 1992

Site-Directed Mutagenesis of Glutathione S-Transferase YaYa: Functional Studies of Histidine, Cysteine, and Tryptophan Mutants Regina W. Wang, *Al Deborah J. Newton,*

Cecil B. Pickett,?

and Anthony

Y. H. Lu*

*Department of Animal and Exploratory Drug Metabolism, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065; and tMerck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Quebec, Canada H9R4P8

Received March 19, 1992, and in revised form April 17, 1992

The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8,143, and 159), two cysteine residues (at positions 18 and 11!2), and a single tryptophan residue (at position 21). Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits. The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively. Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (Iz,.JK,,J toward 1-chloro-2,4-dinitrobenzene and glutathione. Lower but significant k,,JK, values were observed for H159V and ClSA toward 1-chloro-2,4-dinitrobenzene. Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme. These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer. 0 1992

Academic

Press,

ligand binding of GST, various investigators have employed chemical modification, affinity labeling, and GSH analogs to elucidate the possible involvement of various amino acid residues in these events (8-20). From these studies, it has been suggested that cysteine, histidine, tryptophan, aspartic acid, and arginine are present either in the active site or in the vicinity of the active site and that these amino acid residues may play essential roles in catalysis. Recently, site-directed mutagenesis has been used to investigate the functional role of certain amino acids in GSTs (21-27). In a previous study (21), we reported that histidine residues (at positions 8, 143, and 159) in the GST Ya3 homodimer are not essential for catalytic activity, although histidine 159 may be critical in maintaining the proper conformation of this enzyme. In the present study, the histidine mutants of GST YaYa were further characterized in terms of thermal stability and other properties. In addition, cysteine and tryptophan mutants were constructed and characterized. Our results clearly demonstrate that histidine, cysteine, and tryptophan in GST YaYa are not essential for catalysis.

Inc.

MATERIALS

Glutathione S-transferases (EC 2.5.1.18) consist of a family of enzymes which catalyze the conjugation of glutathione to a variety of electrophiles (l-7). In addition, GST2 binds to a large number of hydrophobic compounds, including heme, bilirubin, and polycyclic hydrocarbons. In order to understand the mechanism of catalysis and 1 To whom correspondence should be addressed. ’ Abbreviations used: SDS, sodium dodecyl sulfate; GST, glutathione S-transferase; GSH, glutathione; CDNB, 1-chloro-2,4-dinitrobenzene; PCR, polymerase chain reaction. 86

AND

METHODS

Materials. Reagents used in the present study were obtained from the following sources: restriction endonucleases Sal1 and EcoRI, and T4DNA ligase from Bethesda Research Laboratories; DNA sequencing kits from United States Biochemicak GeneAmp kits from Perkin-Elmer Cetus; deoxyadenosine 5’-a-[%]thiotriphosphate and ?-labeled protein A from Amersham; GSH, CDNB, S-hexylglutathione, S-hexylglutathione-epoxy-agarose, and hematin from Sigma; and A5-androstene3,17-dione from Steraloids, Inc. Oligonucleotide primer design and synthesis. The oligonucleotide primers were synthesized on a Cyclone DNA synthesizer (Biosearch, 3 Subunit

Ya is equivalent

to subunit 1, the proposed nomenclature

(28). 0003-9861/92

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

HISTIDINE,

CYSTEINE,

AND

TRYPTOPHAN

MUTANTS

RESULTS AND DISCUSSION Construction of mutants by site-directed mutagenesis. The rat GST Ya subunit contains three histidine residues at positions 8,143, and 159, two cysteine residues at positions 18 and 112, and a single tryptophan residue at position 21 (29). These residues were selected for sitedirected mutagenesis because of their possible involvement in GST catalysis as suggested by other investigators. Awasthi et al., for example, demonstrated that diethylpyrocarbonate caused a concentration-dependent inactivation of human liver GST+ (10). Complete inactivation was achieved with the modification of one histidine per subunit and inactivation was prevented by glutathione. Also, GST activity is sensitive to sulfhydryl reagents, suggesting that cysteine may play an important role in catalysis (12-14,16-20). In addition, cysteine residues in GST are often the targets for alkylation by quinones and other agents (11, 16, 19, 45). Moreover, chemical modification studies by Van Ommen et al. (14) suggested the presence of a tryptophan residue near the active site of rat GST 4-4.

Enzyme assays. The standard assay for CDNB-conjugating activities was measured spectrophotometrically at pH 6.5 with 1 mM CDNB and 1 mM GSH as described (39). 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 fits of the data to the Michaelis-Menten equation. The inhibition of CDNB-conjugating activities by hematin was conducted at a saturating concentration of GSH (2 mM) and a nonsaturating CDNB concentration

TABLE

Primer

Sense”

Location*

A

Forward

5’-end

B

Inverse

3’.end

Forward Inverse Forward Inverse Forward Inverse

80-102 80-102 364-387 364-387 go-111 91-112

87

YaYa

The intrinsic fluorescence spectra Fluorescence quenching titration. of the purified enzymes were recorded on an SLM 4800 Aminco fluorometer with the excitation wavelength at 282 nm and emission wavelength from 300 to 400 nm. A stock solution of hematin was prepared as described (42, 43), and the concentration determined from the absorbance (400 nm) of a dilution of the stock solution in 40% dimethyl sulfoxide (c = 180 mM-’ cm-‘) (44). Fractional quenching of the intrinsic protein fluorescence upon addition of hematin was measured at hereitat,,,” = 282 nm and Xemisaion= 330 nm. Hyperbolic saturation curves were constructed by plotting the fractional quenching versus the concentration of hematin added to the sample. From these curves, the hematin concentration which caused 50% fluorescence quenching of the enzymes was determined.

PCR mutagenesis. Polymerase chain reactions (PCR) were carried out in 100 pl volume with Taq polymerase as described in the GeneAmp kit (Perkin-Elmer Cetus). For replacement of cysteine 18, cysteine 112, and tryptophan 21 with the desired amino acids, the procedure described by Higuchi et al. (32) was performed with some modifications. The primer pairs (primer A plus inverse mutagenic primer and primer B plus forward mutagenic primer) were used to generate two primary PCR fragments. The two primary PCR fragments were then subjected to the secondary PCR to produce the mutated cDNA fragments as previously described (21). The cDNA fragments were digested with EcoRI and Sal1 and inserted into the expression vector pKK 2.7 (33,34) which was prepared by EcoRI and Sal1 digestion. The constructed plasmids were used to transform Escherichiu coli strain AB 1899 (the lo& protease-deficient strain; E. coli, Genetic Stock Center, Yale University) by the calcium chloride procedure (35, 36). Positive colonies were selected by ampicillin resistance and PCR screening as previously described (21). The DNA sequence analysis for the mutated cDNA was performed as described in the Sequenase kit (United States Biochemical Corp.) with double-stranded plasmid DNA as template (33, 37), with deoxyadenosine 5’-a-[s5S]thiotriphosphate, and with oligonucleotide primers complementary to various regions of cDNA. The histidine mutants at positions 8, 143, and 159 used in this study were the same as those previously prepared (21). Purification of the expressed enzymes. Cells from 1 liter of E. coli overnight cultures which expressed the wild-type or mutant enzymes were harvested and disrupted as described (21, 33). The enzymes were purified by S-hexylglutathione affinity chromatography (33, 38).

Primers

S-TRANSFERASE

(0.4 mM). Isomerase activity was measured with A5-androstene-3,17dione as substrate at pH 8.5 (40). Protein concentration was determined with bovine serum albumin as standard (41).

Inc.). Primer A and Primer B are the 5’-end and 3’-end sequences of Ya cDNA (29,30) with addedEcoR1 and Sal1 restriction sites, respectively. The forward and inverse mutagenic primers with the mismatched codons to the wild-type cDNA were synthesized and are listed in Table I. The cysteine and tryptophan codons were replaced with an alanine codon, GCT, and a phenylalanine codon, TTG, respectively. These codon selections for the specific amino acid were based on the highest codon frequencies found in bacteria (31).

Oligonucleotide

OF GLUTATHIONE

I

Used for PCR Mutagenesis Sequence”

Amino acid changed

5’-ACACCGAATTCATGTCTGGGAAGCCAGTGCT-3’ EcoRI 3’-AGGTCGTCGACCTAAAACTTGAAAACCTTCCTTG-5’ Sal1 5’-GCAGAATGGAG-ATCCGGTGG-3’ 3’~CGTCTTACCTCCGATAGGCCACC-5’ 5’-TTGGTAATAWCCCCCAGACCAA-3’ 3’-AACCATTATCGAGGGGGTCTGGTT-5’ 5’.GTGCATCCGGTTCCTCCTGGCT-3’ 3’-ACGTAGGCCAAGGAGGACCGAC-5’

’ Primer sense is in relation to the cDNA coding strand. * The location refers to the nucleotide position designated by Pickett et al. (29). ’ The oligonucleotides containing the mismatches to wild type are shown with the mismatched

codons underlined.

Cys 18 + Ala Cys 112 *

Ala

Trp 21 + Phe

88

WANG TABLE

Enzyme Activity

Enzyme

of Purified

better than the wild-type GST except for H159V and C18A. Lower k,,,/K,,, toward CDNB for H159V was due to the increase in K,,, (about twofold) and a decrease in kcat. As for ClBA, decreased catalytic efficiency for CDNB and glutathione was primarily due to the decrease in kcat. These results clearly demonstrate that all three histidines, the two cysteines, and the tryptophan in GST YaYa are not essential for catalysis, consistent with recent reports on other GST site-directed mutagenesis studies (22-25). To our knowledge, the role of tryptophan in catalysis has not been studied by site-directed mutagenesis. Thermal stability. In our previous studies (21), we noted that replacement of histidine at position 159 with different amino acids gave variable results. While H159N, H159Y, and H159V retained either full or partial activity, H159K was virtually inactive, suggesting that histidine 159 might be critical in maintaining the proper conformation of this enzyme. To examine this possibility, the heat stability of the purified enzymes was determined by analyzing their CDNB-conjugating activities at elevated temperatures. Figure 1 shows histidine mutant HBV to be slightly less stable than the wild-type enzyme at 60°C. However, after a 15-min incubation at this temperature, mutant H143V lost most of its enzyme activity while mutants H159V and H159N were completely inactivated. Additional studies were carried out by incubating these enzymes for 15 min at 37,46, and 55°C to determine the effect of temperature on their stability (Fig. 2). The enzyme activity of H159V markedly decreased (approximately 50%) at 37”C, thus indicating lower thermostability of this mutant enzyme. Mutants Hl43V and H159N, on the other hand, were stable up to 46°C. However, at 55°C the enzyme activities of these mutants dropped dramatically while the wild-type GST remained active. These data demonstrate that the mutant enzymes, especially

II

Wild-Type

and Mutant

CDNB-conjugating activity (% of wild type)”

Wild type Cysteine mutant C18A C112A Tryptophan mutant W21F

ET AL.

Enzymes

Isomerase activity (% of wild type)”

100

100

55 102

39 137

90

90

’ Conjugating and isomerase activities of purified wild-type 26.3 and 1.22 pmol/min/mg protein, respectively.

GST were

The oligonucleotide primers used for construction of cysteine and tryptophan mutants are shown in Table I. The mutants for the replacement of histidine were generated as previously published (21). The entire cDNA encoding GST YaYa was sequenced in order to confirm the desired base substitutions and to ensure that no other mutations were introduced into the nucleotide sequence during the PCR procedures. Kinetic parameters. As previously reported by our laboratory (21), the histidine mutants of GST YaYa retained either full activity or partial activity depending on the amino acid substitutions. Similarly, cysteine mutants C18A and C112A and tryptophan mutant W21F also exhibited conjugation and isomerase activities (Table II). To further characterize these mutant enzymes, kinetic parameters for the conjugation reaction between CDNB and glutathione were determined (Table III). The catalytic efficiencies (k,,,/K,,,) toward CDNB and glutathione for most of the mutant enzymes were either similar to or

TABLE Kinetic

Constantsa

III

for Wild-Type

and Mutant

Enzymes GSH

CDNB K2 (mM)

Enzyme Wild type Histidine mutant HSV H143V H159V H159N Cysteine mutant ClSA C112A Tryptophan mutant W21F ’ Kinetic

constants

for CDNB

kc, /Km (mM-’ s-l)

kc., /Km (mM-’ s-l)

0.56 f 0.06

52 f 2

0.47 0.71 1.32 0.46

64 78 37 67

92f

6

0.31 + 0.03

51 f 2

164 + 12

55 57 26 61

zk 1 + 3 i 1 f 2

245 ? 19 242 rt 35 95f 7 239 * 22

1 1 1 7

135 -+ 21 111 * 10 28i 2 144 f 28

0.22 0.24 0.27 0.26

0.61 f 0.09 0.43 + 0.11

28 f 2 52 + 5

47f 5 122 f 21

0.30 f 0.03 0.25 f 0.03

21 f 1 52 -c 2

712 5 208 _+ 19

0.73 f 0.17

47 +- 5

0.37 + 0.10

62 + 6

167 f 31

f 0.11 f 0.11 f 0.20 I? 0.14

+ f L +

642

9

and GSH were obtained as described under Materials

f f + f

and Methods.

0.02 0.04 0.03 0.03

HISTIDINE,

0

CYSTEINE,

15

AND

30 45 Time (min)

TRYPTOPHAN

MUTANTS

OF GLUTATHIONE

0

60

S-TRANSFERASE

15

89

YaYa

30 45 Time (min)

60

FIG. 1. Thermal stability of GST wild-type and histidine mutants. Purified wild-type GST, H8V, Hl43V, H159V, and H159N were incubated at 60°C. At indicated time points, aliquots were taken for determination of CDNB-conjugating activities.

FIG. 3. Thermal stability of GST wild-type and cysteine mutants. Purified wild-type GST, C18A, and C112A were incubated at 60°C. At indicated time points, aliquots were taken for determination of CDNBconjugating activities.

H159V, are more easily inactivated by heat than the wildtype enzyme. Their instability is probably caused by structural changes that might have occurred upon amino acid substitution. When C18A and C112A mutants were incubated at 6O”C, these mutants behaved similarly to the wild-type enzyme (Fig. 3). Binding of hematin. Although the histidine, cysteine, and tryptophan mutant enzymes maintained catalytic activities, studies were carried out to determine if such changes altered the ability of these enzymes to bind heme. The fluorescence spectra of wild-type and mutant enzymes are shown in Fig. 4 with the exception of W21F. The overall spectra of the wild-type and mutant enzymes were very similar, i.e., the excitation wavelength at 282 nm and emission maximum at 320-330 nm. However, significant

changes in fluorescence intensities were noted for different mutant enzymes. For example, all histidine mutants exhibited stronger intrinsic fluorescence (Fig. 4A), in comparison to the wild-type enzyme, presumably due to changes in protein conformation. Likewise, the fluorescence intensity of cysteine mutant C112A was also slightly greater than the wild type (Fig. 4B). In contrast, replacement of the only tryptophan in the protein with phenylalanine resulted in much less intrinsic fluorescence. Furthermore, a shift in the maximum spectral peak was noted, resulting in a shorter wavelength for W21F than that observed with any other enzyme (Fig. 4C). The binding of hematin to GST by fluorescence quenching has been studied in several laboratories (46, 47). In the present study, we used this method to determine the heme concentration required to give 50% fluo-

TABLE Binding

of Hematin

to GST Wild-Type

Enzyme

-0

15

30 Temp “C

45

60

FIG. 2. Effect of temperature on the stability of GST and histidine mutants. Purified wild-type GST, H143V, H159V, and H159N were incubated at 37,46, and 55°C for 15 min. CDNB-conjugating activities were determined at the end of incubation.

IV

Heme concentration give 50% fluorescence

concentrations

Enzymes” (PM) to quenching

0.24 31 0.04

Wild type Histidine mutant H8V H143V H159V H159N Cysteine mutant C18A C112A Tryptophan mutant W21F a Hematin 0.3 WM.

and Mutant

0.37 0.65 0.21 0.52

+ h + +

0.04 0.08 0.01 0.06

0.26 k 0.02 0.45 F 0.04 0.27 If- 0.03 used, O-2

pM;

wild-type

or mutated GST,

90

WANG

ET AL.

1.000

Emission Wavelength (nm)

Emission Wavelength (nm)

1.000 c

-

Emission Wavelength (nm)

FIG. 4. Fluorescent spectra of wild-type and mutant enzymes. Fluorescent emission spectra were recorded on an Aminco SLM 4800 fluorometer with the excitation wavelength at 282 nm. Purified enzymes at a concentration of 0.5 pM in 50 mM NaPO,, pH 7.4, were used. (A) Wild type, H8V, H143V, H159V, and H159N; (B) wild type, ClSA, and Cll2A; (c) wild type and W21F.

rescence quenching of the wild-type and mutant enzymes. As shown in Table IV, there were no drastic changes in fluorescence quenching, indicating that all mutants were capable of heme binding. The results of W21F were of special interest since the single tryptophan residue in the GST Ya subunit could play either a direct or an indirect role in heme binding. To further examine its possible role in heme binding to the GST YaYa homodimer, the CDNB-conjugating activity of W21F was studied in the presence of hematin. Enzyme activity was inhibited by hematin for both the wild-type enzyme and W21F mutant in a parallel pattern (Fig. 5). These results clearly indicate that tryptophan, as well as histidine and cysteine, is not the residue responsible for heme binding to GST. In conclusion, despite the fact that chemical modification studies suggest the presence of histidine, cysteine, and tryptophan, at or in the vicinity of the active site, our site-directed mutagenesis studies clearly indicate that these amino acids do not play an essential role in catalysis, nor are they involved in heme binding. However, some of the mutants may have slightly different conformation as judged by their thermal stability and/or fluorescence

[Heme] pM FIG. 5. Inhibition of enzyme activities of purified wild-type and W21F mutant enzymes by hematin. The CDNB-conjugating activities were determined in the presence of 0.02-0.1 pM of hematin in the assay mixture which contained 2 mM GSH, 0.4 mM CDNB, and 0.36 pg of purified wild-type enzyme (0) and W21F mutant enzyme (A).

HISTIDINE,

CYSTEINE,

AND

TRYPTOPHAN

MUTANTS

spectra. The recently published three-dimensional structure of Class ir GST from pig lung (48), coupled with additional site-directed mutagenesis studies, will contribute to our eventual understanding of the mechanism of GST catalysis and ligand binding. ACKNOWLEDGMENTS We thank Drs. Su-Er Wu Huskey, David Linemeyer, and Styliani Vincent for their invaluable advice and discussions, and Mrs. Terry Rafferty for her assistance in the preparation of the manuscript.

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