©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human -Glutamyl Transpeptidase Mutants Involving Conserved Aspartate Residues and the Unique Cysteine Residue of the Light Subunit (*)

Yoshitaka Ikeda , Junichi Fujii (1), Naoyuki Taniguchi (1), Alton Meister (§)

From the (1) Department of Biochemistry, Cornell University Medical College, New York, New York 10021 and the Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mutant human -glutamyl transpeptidases with amino acid substitutions on the light subunit at the Asp residues conserved among several species, and at the unique cysteine residue (Cys-454), were prepared and expressed in a baculovirus insect cell system. Replacement of Asp-423 by Ala or Glu led to major loss of enzyme activity, consistent with the conclusion that Asp-423 is essential for activity. A mutant in which Cys-454 was replaced by Ala was fully active, indicating that the unique light subunit thiol is not required for catalysis. Kinetic analysis of the hydrolysis reaction of L--glutamyl-p-nitroanilide indicated that the decreased activity of Asp-423 mutants is the consequence of an extremely high substrate K value, which is more than a 1000-fold greater than that for the wild-type enzyme, whereas the V is decreased only less than 90-fold. The results suggest that Asp-423, and to a lesser extent Asp-422, interact electrostatically with the -amino group of the -glutamyl donor substrate. Although further studies are required to evaluate the possibility that the reaction involves function of a charge (or proton) relay system, the present work suggests that the -glutamyl moiety of the substrate binds electrostatically to specific groups on the enzyme; this facilitates -glutamyl enzyme formation.


INTRODUCTION

-Glutamyl transpeptidase is a membrane-bound heterodimeric glycoprotein that functions in glutathione metabolism (1, 2, 3, 4, 5) . The enzyme catalyzes transfer of the -glutamyl moiety from -glutamyl compounds as well as from glutathione to a number of amino acids and certain dipeptides. Transfer of the -glutamyl moiety to water leads to hydrolysis. It is thought that a -glutamyl enzyme intermediate, in which the -glutamyl group is covalently bound to the enzyme, is involved in the catalytic mechanism. Since formation of such an intermediate is analogous to the acyl enzyme formed by certain proteases, serine or cysteine residues have been considered as possible active site residues (6, 7, 8, 9) . It has been suggested that the catalytic site is located on the light subunit (6, 10) , which is consistent with the finding that the separated light subunit exhibits protease activity (11). It has not been examined directly whether the unique cysteine residue of the light subunit is required for catalysis. There is accumulating evidence suggesting that a hydroxyl group is involved in catalysis (7, 9, 12) . Thr-523 in the rat enzyme has been identified as the residue that binds the potent inhibitor acivicin (12) . Acivicin binds to Ser-405 in the pig enzyme, and to Ser-406 in the human enzyme (13, 14).() Despite efforts to elucidate the chemical basis of the reaction mechanism of -glutamyl transpeptidase, it is still uncertain as to which enzyme group acts as a nucleophile against the carbonyl moiety of the -glutamyl bond.

Szewczuk and Connell (15) suggested that a carboxyl group and a thiol were present at the active center of -glutamyl transpeptidase; Elce (16) emphasized the importance of a carboxyl group and an amino group in the catalytic mechanism. The specific acidic amino acid residue(s), however, have not yet been identified. An Asp residue might constitute part of a charge (or proton) relay system (catalytic triad) (17) . Alternatively, the negative charge of the acidic amino acid residue might function to attract the positive charge (-amino group) of the substrate. Such an important residue is often strictly conserved among different species.

In the present study, we investigated the properties of mutant -glutamyl transpeptidases in which conserved Asp residues on the light subunit are replaced. We also examined directly whether the sole thiol on the light subunit is required for catalysis by preparing a mutant enzyme in which this Cys residue is replaced by Ala.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and DNA modifying enzymes were obtained from New England BioLabs. Oligonucleotide primers were synthesized by Integrated DNA Technologies, Inc. L--Glutamyl-p-nitroanilide, glycylglycine, and other common reagents were purchased from Sigma.

Construction of the Transfer Plasmid

An NcoI-EcoRI 1.8-kilobase pair DNA fragment containing the entire coding region of human -glutamyl transpeptidase (8) was excised from the expression plasmid used previously (18) . The fragment was then ligated to the pBluescript SK+ (Stratagene) in which an NcoI site had been created at the SmaI site of the vector by insertion of an NcoI linker. The NotI-EcoRI fragment prepared from that plasmid was inserted into a pVL1392 transfer vector (Invitrogen). The resulting plasmid was used to generate the recombinant baculovirus.

Site-directed Mutagenesis

Site-directed mutagenesis was carried out using synthetic oligonucleotide primers according to Kunkel (19). The uracil-substituted single stranded DNAs were prepared from Escherichia coli CJ236 transformed by pBluescript KS+ containing a HindIII-BamHI 1.2-kilobase pair fragment at the 5`-coding region or a BamHI-EcoRI 0.8-kilobase pair fragment encoding the 3`-region of the cDNA for human -glutamyl transpeptidase. The uracil templates were used with oligonucleotide primers to generate mutant sequences. The oligonucleotide primers used in this study are given in . Conserved Asp residues of the light subunit were chosen on the basis of sequence identity among rat (8, 20, 21) , human (8, 22, 23) , pig (24) and E. coli(25) -glutamyl transpeptidases, and human -glutamyl transpeptidase-related enzyme (26) . Asp-422 is conserved in all but the last of these; Asp-423 is conserved in all of these. The mutations obtained were confirmed by dideoxy sequencing (27) , as were the entire sequences after mutagenesis. The corresponding regions of wild-type cDNA were replaced by the mutant sequences. The transfer plasmids for the mutant enzymes were prepared similarly to that for the wild-type enzyme and used for transfection.

Cell Culture and General Manipulation of Viruses

Spodoptera frugiperda (Sf) 21 cells were maintained at 27 °C in Grace's insect media (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 3.33 g/liter yeastolate, 3.33 g/liter lactalbumin hydrolysate, and 50 mg/liter gentamicin. Recombinant viruses were manipulated as described (28) .

Preparation of Recombinant Viruses

The purified transfer plasmids containing wild-type or mutant -glutamyl transpeptidase cDNAs (1 µg) were co-transfected into 5 10 Sf21 cells with 10 ng of Auto-grapha californica nuclear polyhedrosis viral DNA (BaculoGold DNA, PharMingen). Transfection experiments were carried out using the Lipofectin (Life Technologies, Inc.) method (29) . Media containing the generated recombinant viruses were collected 5 days after transfection. The recombinant viruses were further amplified to more than 5 10 plaque-forming units/ml.

Expression of Recombinant -Glutamyl Transpeptidases in Insect Cells (30

2 10 Sf21 cells were infected with the recombinant viruses carrying either wild-type or mutant -glutamyl transpeptidases at multiplicity of infection of 4. The infected cells were harvested about 90 h post-infection.

Purification of the Recombinant Enzymes

Sf21 cells producing the recombinant enzymes were pelleted by centrifugation at 4000 g for 20 min. The cells were homogenized in 10 mM sodium phosphate buffer (pH 7.0) with a glass-glass homogenizer and then stirred for 1 h. The homogenates were centrifuged at 20,000 g for 30 min, and the pellets were resuspended in 10 mM sodium phosphate buffer (pH 6.5). The enzymes were extracted from the suspensions with Triton X-100 (1%) and further solubilized by protease treatment for 5 h at room temperature; papain (Sigma) was added at the amount of the total protein content of the extract. The solubilized enzymes were applied on a hydroxylapatite (Bio-Rad) column pre-equilibrated with 10 mM sodium phosphate buffer (pH 6.5) and then eluted with a linear gradient established between 10 mM and 0.5 M buffer. The active fractions were further purified by isoelectric chromatography using PBE 94 and polybuffer 74 (Pharmacia). The enzymes eluted from PBE 94 by use of a pH gradient from pH 7.4 to pH 4.0 were passed through a column of Sephacryl S-200 HR (Pharmacia) equilibrated with phosphate-buffered saline (PBS)() to remove polybuffer.

The enzymes were detected by enzyme activity assay at each chromatographic step for the wild-type and the partially active mutants. An immunological method was used to detect the enzyme fractions of mutants that had very low levels of activity. Fifty µl of each fraction obtained in the chromatography was added to a well of a 96-well microtiter plate (Nunc), and the plate was incubated at room temperature for 1 h to allow attachment of the proteins to the wells. After blocking with 1% bovine serum albumin (in PBS), and washing the plate with Dulbecco's phosphate-buffered saline containing 0.05% Tween 20 (T-PBS) three times, 50 µl of goat anti-human kidney -glutamyl transpeptidase antibody (31) diluted with T-PBS was added to each well. The plate was incubated and washed as described above. Horseradish peroxidase-conjugated rabbit anti-goat IgG antibody (Sigma) was then used as a second antibody. After washing the plate, colors were developed by use of the peroxidase-substrate kit (Bio-Rad), according to the manufacturer's instructions.

Enzyme Immunoassay

Enzyme-linked immunosorbent assay for human -glutamyl transpeptidase was used to estimate the relative expression levels of -glutamyl transpeptidase proteins produced in the insect cells. The assay and analyses were carried out basically as described (18) , using a peroxidase-substrate kit (Bio-Rad) to develop the color.

Electrophoresis

The purified enzymes were subjected to SDS-polyacrylamide gel electrophoresis analysis on 11% gels, according to Laemmli (32) . The proteins were visualized by ammonical silver staining (33) .

Enzyme Activity Assay

Standard assay for -glutamyl transpeptidase activity was performed at 37 °C using 1 mML--glutamyl-p-nitroanilide as a donor substrate and 20 mM glycylglycine as an acceptor in 0.1 M Tris-HCl buffer (pH 8.0) as described (2) . One unit of activity is defined as the quantity of enzyme that releases 1 µmol of p-nitroaniline/min.

Kinetic Analysis

Enzymatic activity for transpeptidation was assayed at 37 °C using 0.25-2 mML--glutamyl-p-nitroanilide and 5-80 mM glycylglycine, as donor and acceptor substrates, respectively, in 0.1 M Tris-HCl buffer (pH 8.0). In assessment of hydrolysis, 6.7 µM to 5 mML--glutamyl-p-nitroanilide was used in the absence of glycylglycine. Kinetic parameters for hydrolysis were calculated using the data of the range in which a substrate activation kinetics did not appear. When the wild-type and mutant enzymes, except Asp-423-substituted mutants, were assayed, 0.02-0.05 µg of the purified enzymes was subjected to each activity determination; 2-10 µg of purified enzymes were used for Asp-423 mutants. The release of p-nitroaniline was monitored at 410 nm using a Cary model 210 spectrophotometer (Varian). In some studies on mutants whose activities were too low to permit continuous monitoring, the release of p-nitroaniline was assessed by end point assay. The reaction rates by enzymes were determined after subtraction of the contribution of the spontaneous hydrolysis of the substrate without an enzyme. Kinetic parameters were calculated using nonlinear regression analysis based on the Marquart algorithm. When transpeptidation, facilitated by glycylglycine as an acceptor substrate, was examined in some mutant enzymes as well as the wild-type, 50 µML--glutamyl-p-nitroanilide was used as a donor substrate without or with 0.25, 0.5, 1.0, 2.0, and 4.0 mM glycylglycine as an acceptor in 0.1 M Tris-HCl buffer (pH 8.0).

Protein Determination

Protein contents were determined by bicinchoninic acid method using bovine serum albumin as the standard (34).


RESULTS

The mutants listed in were expressed in the baculovirus system as described under ``Experimental Procedures.'' Several of these (D390A, D559N, and D559E) were successfully expressed, as indicated by determinations of enzymatic activity of the crude cell extracts. Their activities were 26, 6.0, and 7.0 milliunits/mg cell protein, respectively, whereas activity in noninfected cells as a control was nearly undetectable (0.015 milliunits/mg cell protein). However, although the cell extracts exhibited activity, the corresponding proteins could not be significantly detected by the enzyme-linked immunosorbent assay system (which did not detect less than 1% of the protein formed in extracts of cells expressing the wild-type enzyme). It appears that these mutants are expressed, but at low levels. The low activities observed in the cell extracts appear to be related to low expression level rather than to expression of proteins that have intrinsically low enzymatic activities. Consistent with this interpretation is the finding that mutant D390E (in contrast to mutant D390A, which was very poorly expressed), was significantly expressed (6.6% of wild-type protein expression and 7.3% of wild-type enzyme activity).

The wild-type enzyme and the mutant enzymes that were expressed in sufficient amounts were purified. Each of the purified enzymes was found to exhibit two bands on SDS-polyacrylamide gel electrophoresis that corresponded to M values of 44,000 and 24,000, respectively, for the heavy and light subunits of the enzymes. The specific enzymatic activities of these () were determined by the standard assay (with L--glutamyl-p-nitroanilide and glycylglycine). Replacement of Asp-390 by Glu, and of Cys-454 by Ala, did not lead to decrease of specific activity. Replacement of Asp-422 by Ala or Glu did not lead to major loss of enzymatic activity. In contrast, the mutants in which Asp-423 was replaced by Ala or Glu, and the double mutant (D422E + D423E), exhibited very low enzymatic activities; thus, mutant D423A showed only about 0.001% of the specific activity of the wild-type enzyme. When the mutants in which Asp-423 was replaced were assayed in the presence of glycylglycine, there was no enhancement of the rate of p-nitroaniline formation; such enhancement was found, as expected, with the wild-type enzyme (data not shown). At concentration range higher than 80 µML--glutamyl-p-nitroanilide in the absence of glycylglycine, the wild-type enzyme exhibited a substrate activation kinetics clearly, which does not give a linear curve on the double-reciprocal plot (35) (Fig. 1). L--Glutamyl-p-nitroanilide serves as an acceptor substrate as well as a donor, and autotranspeptidation reaction proceeds much more rapidly than hydrolysis. Nevertheless, this type of kinetics was not observed in Asp-423-substituted mutants, unlike the case of the wild type (Fig. 1). These results showed that these mutants lacked transpeptidation reaction and only catalyze hydrolysis.


Figure 1: Double-reciprocal plots of activity in the absence of an acceptor substrate for the wild-type and Asp-423 mutant human -glutamyl transpeptidases. Activities were assayed at 37 °C in 0.1 M Tris-HCl buffer (pH 8.0), without an acceptor substrate. Concentrations of L--glutamyl-p-nitroanilide used were in the range of 0.08-5 mM. The panel of the wild type also includes an inset showing the values obtained at concentrations less than 0.08 mM.



Kinetic parameters for transpeptidation were determined for the enzymes purified from several mutants and from the wild type (I), according to the following equation,

On-line formulae not verified for accuracy

The catalytic properties of D390E and of C454A were similar to those of the wild type (Tables II and III). The two substitutions made for Asp-422 led to decreased V values for transpeptidation. Mutant D422A had somewhat increased K value for L--glutamyl-p-nitroanilide and increased K for glycylglycine as compared with the enzyme isolated from mutant D422E. These observations suggest that Asp-422 may interact with the donor substrate in the -glutamyl binding site.

Determinations of the kinetic parameters for hydrolysis () illustrate the expected decrease in V for D423A (which is significantly less than found for D423E) and also show that the loss of a negative charge (D422A) increases K. On the other hand, replacement of Asp-422 by Glu did not increase the K as much as did replacement by Ala. Thus, it appears that the negative charge at Asp-422, although not required for catalysis, may be involved in substrate binding.

Although replacement of Asp-423 leads to decrease in V, it produces a dramatic increase in K to values that are a 1000-fold or higher than that of the wild type. Unlike the effect of Glu substitution at Asp-422, substitution of Glu for Asp-423 increases V and K (as compared with the effect of Ala substitution); possibly the effect is steric, related to the more bulky Glu moiety, which may offset the electrostatic effect. It is interesting to note that the double Glu mutant (D422E + D423E) has a lower K than D423E consistent with occurrence of interaction between neighboring carboxyl groups.


DISCUSSION

These studies constitute a further effort to identify the functional amino acid residues at the active center of this enzyme, whose three-dimensional structure is still unknown. Such information will be valuable for interpretation of crystallographic studies, some of which are now in progress, on the -glutamyl transpeptidase of E. coli(36) . Amino acid residues that are thought to be located at or near the active site include Thr-523 (rat (12) ), Ser-405 (pig (14) ), Ser-406 (human (14) ), and several residues of the heavy subunit (rat (37) , human (18) ). Data suggesting the participation in catalysis of a hydroxyl group (7, 9) , an amino group (16) , a carboxyl group (15, 16) , a cysteine residue (8, 15, 16) , a histidine residue (4), and an arginine residue (18, 37, 38) have been published. The present studies show that Cys-454 (equivalent to Cys-453 in the rat enzyme), which is the sole thiol on the enzyme's light subunit, is not required for catalysis. Thus, the human enzyme mutant C454A is fully active catalytically (). This cysteine residue is conserved among the mammalian -glutamyl transpeptidases whose primary structures are known (8, 20, 21, 22, 23, 24, 39) . Interestingly, the known bacterial enzymes have serine residues at the corresponding positions (25, 40). It cannot be excluded that this cysteine residue has a regulatory or other function in the mammalian enzymes which is not required in the bacterial enzymes.

The studies reported here indicate that, in contrast to Cys-454, Asp-423 is an important active site residue.() Its replacement by Ala or Glu led to marked decreases in specific activity (). Nevertheless, V (hydrolysis) values were about 1% of the wild type (), but there was more than a 1000-fold increase in K. Such data do not seem to reflect operation of a charge (or proton) relay system (Ser-His-Asp) (17) , where one might expect a much greater effect of amino acid replacement on V(41, 42, 43) . The evidence given above suggests that Asp-422 also participates, together with Asp-423, in substrate binding rather than in catalysis, although Asp-423 is clearly more important. The findings suggest that these adjacent residues (Asp-422 and Asp-423) perform essentially the same function, i.e. to anchor the amino group of the -glutamyl amino acid substrate to the enzyme. It has been suggested that a conformational change may occur after formation of the -glutamyl enzyme which facilitates binding of the acceptor substrate (44) . Such a conformational change may not be possible, or be hindered, in the mutants that lack Asp-423, thus accounting for the observed decrease in the transpeptidase activity exhibited by them. It seems notable that a similar loss of transpeptidase activity was previously found in a mutant in which Arg-107 was replaced (18) ; this residue is thought to interact with the carboxyl group of the -glutamyl amino acid substrate. Accordingly it would seem that both the -amino group and the -carboxyl group of the -glutamyl donor may attach to electrostatic binding sites and that such binding may favor orientation of the -carboxyl moiety so that it can interact with the -glutamyl binding site (probably a hydroxyl group) on the enzyme (Fig. 2).


Figure 2: Possible role of Asp-423 in human -glutamyl transpeptidase.



  
Table: Oligonucleotide primers used for mutagenesis and designations of mutants


  
Table: Specific activity of purified -glutamyl transpeptidases


  
Table: Kinetic parameters for wild-type and mutant -glutamyl transpeptidases in transpeptidation


  
Table: Kinetic parameters for wild-type and mutant -glutamyl transpeptidases in hydrolysis (in 0.1 M Tris-HCl buffer (pH 8.0) at 37 °C)



FOOTNOTES

*
This research was supported in part by National Institutes of Health Grant 2 R37 DK12034 from the United States Public Health Service (NIDDK) and by grants-in-aid for cancer research and scientific research on priority areas from the Ministry of Education, Science, and Culture, Japan. 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.

§
Deceased April 6, 1995.

The numbering of the amino acid residues of the human and rat -glutamyl transpeptidases differ by one residue. Thus, Asp-422 and Cys-453 in the rat enzyme are equivalent, respectively, to Asp-423 and Cys-454 in the human enzyme.

The abbreviation used is: PBS, phosphate-buffered saline.

Independent studies in this laboratory on the chemical modification of active site residues in rat kidney -glutamyl transpeptidase showed that inactivation of the enzyme by treatment with iodoacetamide leads to esterification of Asp-422 (equivalent to Asp-423 in the human enzyme (45)).


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