THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc

Vol. 259, No. 2, Issue of January 25, pp. 1043-1050,1984 Printed in U.S.A.

Mechanism of Selenium-Glutathione Peroxidase and Its Inhibition by Mercaptocarboxylic Acids and Other Mercaptans*

(Received for publication, May 23, 1983)

Jean Chaudiere, Eric C. Wilhelmsen, and A1 L. TappelS From the Department of Food Science and Technology, Uniuersity of California, Dauis, Californin 95616

In a systematic search for effectors of glutathione peroxidase, a number of mercaptocarboxylic acids and tertiary mercaptans were found to be strong and spe- cific inhibitors of the enzyme glutathione peroxidase. Assessment of various models was made by linear and nonlinear least squares fitting techniques. The results support the formation of reversible enzyme-inhibitor complexes. The active site selenium is trapped by the rapid binding of the inhibitor in competition with GSH. Data are consistent with the formation of thioselenen- ate adducts of the active site. The kinetic model which best describes the observed inhibition by the very strong inhibitor mercaptosuccinate implies that a se- lenenic acid with a kinetically‘significant lifetime is not formed when hydroperoxide is reduced. A nonco- valent binding site for GSH or the presence of a cys- teine residue at the active site of the enzyme provides a mechanistic rationale for the observed kinetics. Three of the most potent inhibitors found in this study, mercaptosuccinate, penicillamine, and a-mercaptopro- pionylglycine, are currently used as slow-acting drugs in the treatment of rheumatoid arthritis. Overall, the evidence suggests t,hat glutathione peroxidase may be involved in the etiology of this disease.

Using GSH as the reducing equivalent, glutathione peroxi- dase (GSH:H202 oxidoreductase, EC 1.11.1.9) reduces hydro- gen peroxide to water (1) and organic hydroperoxides to the corresponding alcohols (2). The enzyme contains stoichio- metric amounts of selenium (3, 4) and has been thought to account for the essentiality of selenium as a nutrient (5). Selenium in glutathione peroxidase has been shown to be in the form of selenocysteine (6, 7). This amino acid residue is within the polypeptide backbone (8, 9), and it is generally agreed that it is a major component of the active site (5 , 10, 11). In recent years, various selenoproteins have been identi- fied in animal tissues (12-14), suggesting the existence of selenoenzymes other than glutathione peroxidase. Under- standing the chemistry of selenium at the active site of glutathione peroxidase may help to elucidate some general features of selenocysteine-mediated catalysis.

The kinetic data obser+ed for hamster liver glutathione peroxidase, which was used in this study, are compatible with the ter uni ping-pong mechanism reported for the enzyme

* This investigation was supported by Research Grant AM-06424 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. 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 should be addressed.

from other various sources (15). Photoelectron spectroscopy (16) and the carboxymethylation of the active site (6, 15) support the existence of a selenocysteine selenolate as the resting form of the enzyme at physiological concentrations of GSH. However, the oxidized forms of the active site selenium during catalysis are controversial (17). Steady state kinetics do not separate the bimolecular rate constants for the two consecutive reactions of the oxidized forms of glutathione peroxidase with GSH in the absence of a specific effector of the enzyme. Inhibitors that interact with a given form of the enzyme may prove to be a useful tool to characterize the steps of the enzyme cycle.

Reversible enzyme inhibitors can be studied by conven- tional kinetics in more detail than irreversible effectors. Coen- zyme A is the only compound reported to reversibly inhibit glutathione peroxidase a t low concentration (18). The inhi- bition by coenzyme A is not well understood, but the sulf’hy- dry1 group and the nucleotidic moiety of the molecule seemed to be required for inhibition of glutathione peroxidase. Other reversible effectors of glutathione peroxidase include pyrimi- dine nucleotides, which were reported to behave as uncom- petitive inhibitors of the enzyme from pig red blood cells (19). A few GSH analogs act as weak reducing substrates in the absence of GSH (20), and many mercaptans, such as mercap- toethanol, will compete for GSH in the second reduction step of the enzyme cycle (21). A necessary reductive preactivation of the enzyme can also be performed by many mercaptans (22).

In practice, the above mentioned compounds have not afforded great insights into the catalytic cycle of glutathione peroxidase. The studies of these compounds strongly suggest, however, that a search for specific and reversible effectors of the active site of glutathione peroxidase should focus on. bi- or polyfunctional mercaptans. In making such a search, we discovered that mercaptocarboxylic acids and tertiary mer- captans are potent inhibitors of glutathione peroxidase. Herein is reported a detailed study of the inhibition by these mercaptans that provides new insight into the mechanism of glutathione peroxidase. A possible pharmacological signifi- cance of this inhibition is also briefly discussed.

MATERIALS AND METHODS

Chernica&-”ll mercaptans were >98% pure and were obtained from commercial sources. Mercaptosuccinate and 8-mercaptovaline used for kinetic analyses were purchased from Sigma.

S-Methylmercaptosuccinate was prepared by reaction of 10 mM sodium thiomalate with a 3-fold excess of methyl iodide in 0.1 M borate buffer, pH 9.0, until the sulfhydryl group was no longer detected with Ellman’s reagent (23). Excess methyl iodide was then hydrolyzed by adjusting the pH to 12 with sodium hydroxide.

Preparation and Assays of Glutathione Peroxidase-Gutathione peroxidase used in this study was highly purified (approximately 160- fold) from hamster liver as described elsewhere (15). The preparation was devoid of selenium-independent glutathione peroxidase activity

1043

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1044 Mechanism and Inhibition of Glutathione Peroxidase

and had a specific activity of 500 pmol of t-butylhydroperoxide reduced/min and per pmol of selenium at pH 7.6,37 "C, and 0.25 mM GSH. A highly concentrated solution of the enzyme was stored at, 4 "C in 50 mM Tris-HC1, 10% ethanol, p H 7.7. Initial rates of glutathione peroxidase activity were monitored at 37 "C with a ther- mostated spectrophotometer using a glutathione reductase-coupled assay (24). The decrease in NADPH absorbance a t 340 nm was monitored for at least 2 min. Unless otherwise mentioned, initial concentrations of reactants in the cuvette were as follows: 0.25 mM GSH, 0.20 mM t-butylhydroperoxide (initiator), 0.12 mM NADPH, 1 unit/ml of glutathione reductase, 3.0 nM glutathione peroxidase, 50 mM Tris-HC1, 0.1 mM EDTA, pH 7.7. The mixed components, with the exception of hydroperoxide, were preincubated at 37 "C for a t least 5 min.

For studying the structural requirements for inhibition, the above assay components were used in the presence of 0.2 mM effector. As a control of the validity of the assay, initial rates of glutathione reduc- tase activity were measured in the presence of each effector. The decrease in NADPH absorbance was monitored for at least 2 min at 340 nm and 37 "C, with the following concentrations of reactants in the cuvette: 0.25 mM GSH, 0.20 mM GSSG (initiator), 0.20 mM NADPH, 0.03 unit/ml of glutathione reductase, and 50 mM Tris-HC1, 0.1 mM EDTA, p H 7.7.

Steady State Kinetics of Inhibition-The p H profiles of glutathione peroxidase activity with and without inhibitor were obtained using a three-component polybuffer. The buffer, containing 50 mM each of Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-l,3- diol, and borate, was titrated to pH with either sodium hydroxide or hydrochloric acid. Rate equations fitting various hypothetical mech- anisms of inhibition were derived by the use of the King-Altman method (25). In order to assess the validity of each of these mecha- nisms, glutathione peroxidase activity was monitored a t a limiting concentration of t-butylhydroperoxide a t either constant GSH con- centration and variable inhibitor or vice versa. In each reaction system, glutathione peroxidase activity was initiated with approxi- mately 45 pM t-butylhydroperoxide in the presence of 80 p M NADPH and 1 unit/ml of glutathione reductase. Absorbance monitoring was continued for at least 1 min after completion of hydroperoxide reduc- tion. Each curve of absorbance uersus time was digitized with a model 4662 interactive digital plotter of a Tektronix 4051 computer. Time intervals between digitized points were between 3 and 6 s. Linear least squares fits to the digitized data for the integrated rate equation were obtained by the use of a Tektronix 4051. The corresponding BASIC routine included a correction for nonenzymatic reduction of t-butylhydroperoxide by GSH based on a bimolecular rate constant of 2.5 M" min" and measured with the glutathione reductase-coupled assay.

For nonlinear least squares fitting of digitized data to the derived functions, enzyme velocity was calculated along each progress curve using the derivative of a cubic spline fit of 3 knots (26). Each of these fits had a correlation coefficient higher than 0.999 and gave a random distribution of residuals. Hydroperoxide concentration was obtained by difference between the concentration at the time of interest and the final concentration based on the measured NADPH. The resulting data (velocity uers'sus hydroperoxide concentration) were then used for nonlinear least squares fitting ( 2 7 ) to each of the derived functions. For several mechanisms, good estimates of the parameters were required to obtain convergence in the nonlinear least squares pro- gram. These estimates were checked using a grid search technique as recommended by Bevington (28) when there was some doubt as to whether there was a better solution. In the grid search, the parameters were varied over five orders of magnitude by powers of three. For a few equations, a Simplex algorithm (29) was used to improve the estimates obtained from the grid search.

Other Enzyme Assays-Selenium-independent glutathione peroxi- dase from rat liver was purified to remove the selenoenzyme as previously described (30). Its glutathione peroxidase activity was measured with cumene hydroperoxide as substrate. Initial rates of other enzyme activities were assayed as described in the literature: catalase from beef liver (31), alcohol dehydrogenase from horse liver (32), and isocitrate dehydrogenase from pig heart (33).

RESULTS AND DISCUSSION

The results described herein were obtained with glutathione peroxidase from hamster liver, however, similar inhibitions were observed with glutathione peroxidase from many other

sources and no exceptions were found. Since a rigorous proof cannot be obtained from kinetics regarding the chemistry of a reaction, it is important to bear in mind that our interpre- tations are generally supported by several lines of evidence as indicated below.

Specificity of Inhibitor Interaction-Control assays of glu- tathione reductase in the presence of each effector used in this study supported the validity of the glutathione reductase- coupled assay for glutathione peroxidase. For a 33-fold dilu- tion of the reductase concentration used in the peroxidase assay, a significant deviation from control values of reductase activity was observed with only three of the effectors. These values were 14, 88, and 60% of the control activity with methyl-3-mercaptopropionate, P-mercaptopyruvate, and t-bu- tylmercaptan, respectively. Even in the presence of these three compounds, where some deviation from control values was observed, the reductase activity was still in large excess as shown by the fact that decreasing the level of coupling reductase by a factor of two in the control did not affect measured initial rates.

The effect of various mercaptans on glutathione peroxidase activity is shown in Fig. 1. Cysteine and mercaptoethanol caused only moderate inhibition at or below 0.1 mM. This slight inhibition is representative of the trend observed with many mercaptans, including simple thiols such as mercapto- ethane, aminothiols such as mercaptoethylamine, and nonvi- cinal dithiols such as dithiothreitol and lipoate (data not shown). This inhibition pattern has been reported (21) and can be explained by the fact that the second reduction step in the glutathione peroxidase cycle is only moderately specific for GSH (21).

More potent inhibition was observed with coenzyme A at low concentrations. The a-mercaptocarboxylic acids, Z-mer- captopropionate (thiolactate) and mercaptoacetate (thiogly- colate), led to apparent inhibitions that were, respectively, similar to and stronger than those caused by coenzyme A. A much more potent inhibition was observed with the @-mer- captocarboxylic acids, 2-mercaptobenzoate (thiosalicylate) and mercaptosuccinate (thiomalate). In mercaptosuccinate, which is the most potent inhibitor in the series, the sulfhydryl group is a to a carboxylate group and /3 to another carboxylate group.

Many mercaptans and their analogs were used at 0.2 mM to analyze the structural features of potent inhibitors (Table I). Generally, a sulfhydryl group in /3 position to a carboxylate group leads to optimal inhibition. This was observed with mercaptosuccinate, 2,3-dimercaptosuccinate, z-mercaptoben- zoate, /I-mercaptovaline, and P-mercaptopyruvate. @-Mercap- topyruvate is in rapid equilibrium with a dithiane dimer (381, which may explain why less inhibition is observed than with

. 2

EFFECTOR CONCENTRATION (pM) 25 50 75 IO0

FIG. 1. Effect of various thiols on hamster liver glutathione peroxidase activity with t-butylhydroperoxide as substrate. Each data point is the mean value of two independent measurements a t 37 'C and 0.25 mM GSH. Effectors are (A) cysteine, (0) mercap- toethanol, (W) mercaptoacetate, (A) coenzyme A, (V) 2-mercaptopro- pionate, (0) 2-mercaptobenzoate, and (0) mercaptosuccinate.

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Mechanism and Inhibition of Glutathione Peroxidase 1045

Effector (0.2 mM)

2-Mercaptoethanol Cysteine Coenzyme A Mercaptosuccinate 2,3-Dimercaptosuccinate Mercaptoacetate 2-Mercaptopropionate 2-Mercaptohenzoate 3-Mercaptopropionate 0-Mercaptovaline t-Butylmercaptan 0-Mercaptoisoleucine 0-Mercaptopyruvate Methyl(3-mercaptopropio-

Methylmercaptoacetate a-Mercaptopropionylglycine 2-Mercaptoethane sulfonate S-Methylmercaptosuccinate Carboxymethylmercaptosuc-

~-Thiazolidine-4-carhoxylate Malate Succinate

nate)

cinate

TABLE r Effect of mercaptocarboxylic acids and related compounds on

glutathione

a Glutathione Deroxidase at

peroxidase activity

tivitf

I .

I :

73.0 f 4.8 9.44, 9.6 56.0 + 3.3 8.31' 15.2 + 0.8 9.6 0.0 + 0.0 10.37, 10.38 0.0 + 0.0 Not found

36 + 2.5

90.3 + 6.4 10.25' 61.0 + 6.2 9.85', 0.3 + 0.2 10.3' 99.7 + 2.4 10.3' 59.9 + 3.4

Not found 16.9 + 1.7 9.25'. e 73.4 + 3.2 11.22 0.0 + 0.0

10.46, 10.58' 0.5 + 0.3 10.54, 10.80 25.1 + 1.0

9.96 0.8 + 0.6 10.11 17.3 + 0.4 10.11

86.0 + 5.51 I 102.3 + 1.0 105.7 + 8.8

I

L

Reference or sourceb

34,35 36

34,37

37 37 34

34,37 34,37

36 This work

This work This work This work This work

35

d

vitv is exmessed as Dercentages of the I cti I -

control (mean + range of two independent measurements) at 0.25 mM GSH and 37 "C.

* Preference was given to compilations of pK, (SH) values. Most of the numbers are average values computed from original data obtained at 0.1 ionic strength.

Assignment of macroscopic constants of aminothiols is tentative. pK, (SH) neither measurable by titration nor found in literature.

e Titration.

other P-mercaptocarboxylic acids. The much smaller inhibi- tion by cysteine may be explained by a preferential interaction of the sulfhydryl group with the amino group, as occurs with mercaptoethylamine. It is difficult to account for the lack of strong inhibition with 3-mercaptopropionate.

That a sulfhydryl group is essential for inhibition is sup- ported by two lines of evidence. First, analogs of mercapto- succinate that do not bear a sulfur-containing group, such as malate and succinate, are not inhibitors. Second, substitution of an S-alkyl group for a sulfhydryl group also relieves the inhibition. This is illustrated by the effects of S-methylmer- captosuccinate, carboxymethylmercaptosuccinate, and the cyclic t-thiazolidine-4-carboxylate (thioproline). Similarly, the importance of the carboxylate group is supported by the moderate inhibition observed with methyl-3-mercaptopropio- nate and by the lack of inhibition observed with methylmer- captoacetate. That a sulfonate group cannot fulfill the func- tion of the carboxylate group is supported by the weak inhi- bition observed with 2-mercaptoethanesulfonate.

Inspection of pK.(SH) values reported in Table I shows that all inhibitors have rather weakly acidic sulfhydryl groups. A high pK.(SH) is probably not a determinant of the effect because of the lack of a quantitative relationship. The high pK,(SH) values of most mercaptocarboxylic acids may rather reflect a slight tendency to form cyclic structures involving hydrogen bonding between the carboxylate and sulfhydryl groups. If accessibility to such a structure were an important feature of these inhibitors, one would expect P-mercaptocar- boxylic acids to be somewhat stronger inhibitors than their a-isomers because of the greater thermodynamic stability of six-membered rings over five-membered rings. On the other

hand, given its highly hindered position, the sulfhydryl group of P-mercaptovaline should not easily adopt this type of conformation. This suggested that tertiary mercaptans might be a second family of inhibitors. This hypothesis was tested with @-mercaptoisoleucine or t-butylmercaptan. As shown in Table I, t-butylmercaptan was a very potent inhibitor of glutathione peroxidase. However, more hindrance near the sulfhydryl group seems to relieve the inhibition, as shown with @-mercaptoisoleucine. Kinetic studies were not done to establish the mechanism of inhibition by the tertiary mercap- tans in this study.

As discussed below, kinetic data support the assumption that mercaptocarboxylic acids react with an oxidized form of the active site selenocysteine in glutathione peroxidase. The specificity of this type of inhibition is confirmed in Table 11. This table shows that two enzymes that also decompose hydroperoxides (catalase and selenium-independent glutathi- one peroxidase), two essential sulfhydryl enzymes (alcohol dehydrogenase and isocitrate dehydrogenase), and glutathione reductase, which bears an essential dithiol/disulfide bond at its active site, were either not inhibited or only moderately inhibited by a high concentration of either mercaptosuccinate or a-mercaptopropionylglycine. Mercaptosuccinate was se- lected to investigate the kinetics of inhibition of glutathione peroxidase because it could be used at concentrations much lower than the hydroperoxide and because the inhibition apparently reached steady state in a few seconds.

As shown in Fig. 2, the pH profile of glutathione peroxidase

TABLE I1 Effect of mercaptosuccinate and a-mercaptopropionylglycine on

various enzymes All enzyme activities were measured as initial rates at 37 "C and

in the presence of 0.25 mM GSH and 0.2 mM of mercaptosuccinate or a-mercaptopropionylglycine. Catalase activity was corrected for a small nonenzymatic rate due to sulfhydryl compounds.

Enzyme Mercaptosuccinate a-Mercaptopropionyl elvcine "" ~-~

% of control enzyme activity" Selenium-independent 89.4 * 3.8 b

Alcohol dehydrogenase 106.0 f 3.8 102.2 f 4.1 Isocitrate dehydrogenase 106.4 f 4.0 100.1 f 3.0 Glutathione reductase 92.7 f 8.3 97.0 f 3.9 Catalase 96.8 _t 10.0 98.9 f 6.9

glutathione peroxidase

aResults are expressed as mean f S.D. of three independent measurements.. Control enzyme activities were measured in the ah- sence of effector.

* Not done.

PH FIG. 2. Effect of pH on glutathione peroxidase activity with

and without mercaptosuccinate. Each data point is the mean value of two independent measurements at 37 "C and 0.25 mM GSH with t-butylhydroperoxide as substrate. 0, glutathione peroxidase without mercaptosuccinate; 0, glutathione peroxidase @-fold more concentrated) in the presence of 2 p M mercaptosuccinate. Values are expressed as percentage of the optimal enzyme activity in the three- component polybuffer, pH 8.5, without mercaptosuccinate.

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1046 Mechanism and Inhibition of Glutathione Peroxidase

activity with and without mercaptosuccinate indicates that inhibition is not pH-dependent around neutral pH. The range of pH that could be used with the reductase-coupled assay is below the pK,(SH) of mercaptosuccinate. Therefore, the thiolate form of this inhibitor is not required in the mecha- nism of inhibition.

Steady State Kinetics-Studies of steady state kinetics are often the only way to obtain a description of enzyme inhibi- tion without detailed understanding of the chemistry in- volved. With non-Michaelian kinetics or with complex inhi- bition, theoretical models require extensive sets of initial rate data for analysis. The information obtained from progress curves is equivalent to information obtained from initial rate assays when product inhibition is negligible, but much infor- mation is obtained at low substrate concentrations where initial rate measurements are difficult. The information is obtained in fewer assays by following progress curves. Follow- ing progress curves is of particular advantage when the amount of enzyme is limiting.

In this study, interpretation of velocities along entire prog- ress curves was not hampered by product inhibition. Gluta- thione peroxidase initial rates were not affected by up to 15% ethanol, and glutathione reductase prevented the accumula- tion of significant amounts of GSSG. The enzyme mechanism was first assessed in the absence of inhibitor using Models I and I1 on Fig. 3. Model I has generally been considered to be the correct model for glutathione peroxidase (22, 39, 15), but it had not been assessed previously by a nonlinear least squares fitting technique. As shown in Table 111, the reverse step is not substantiated by the kinetic data. The reversible pathway in Model I1 was considered because it might have explained the apparent complexity of the inhibition by mer- captosuccinate.

As mentioned above, the kinetic pattern of hamster liver glutathione peroxidase is similar to the one of the rat liver enzyme. It may be described by the following Dalziel equation:

EoIV = @ I j A + &z/B (1)

where Eo is total enzyme concentration, V is enzyme velocity, A is hydroperoxide concentration, B is GSH concentration, and 41 = l / k l and 42 = (k2 + k3)/(kz X k3), using the second order rate constants defined in Fig. 3. In the conditions of the reductase-coupled assay, B is constant and the following integrated rate equation can be obtained (39):

(EO x t) lP, = @ 1 x [ln(AdAo - Pr)/Prl + @ d B (2)

where t is time, P, is product concentration at time t, and A. is initial hydroperoxide concentration. Since GSH concentra- tion is always constant in the conditions of the coupled assay, the form of a similar integrated rate equation can be deduced from any theoretical mechanism of inhibition.

As shown in Fig. 4, at constant GSH concentration and for various mercaptosuccinate concentrations, the kinetic pattern

I il FIG. 3. Two possible models for glutathione peroxidase

cycle. E is the most reduced form of the enzyme. F is obtained after reduction of the hydroperoxide and release of alcohol. A and B are the concentrations of hydroperoxide and GSH, respectively; and kl , kz, and k3 are bimolecular rate constants, whereas k-2 is monomo- lecular. These vector diagrams were used for King-Altman calcula- tions. Note that enzyme-substrate complexes in addition to E, F, and G are not identifiable by steady state kinetics.

TABLE I11 Statistical evaluation of rate equations used for nonlinear kast

sauares fitting Degrees of freedom in u X Rejection

reduced sF;2 io4 criteriad equation

Sum of Model" Rate equationb

I 1 2 1.2 4.9 Accepted I1 2 3 1.1 4.9 CZ, c,

111 (3) ks = k-6 = 0 3 15 11 c6

IV (3) kr = k-4 = 0 3 7.0 7.3 c1, cg V (3) kg = k-4 = 0 4 4.2 5.7 c3

VI (3) k4 = k-6 = 0 4 6.4 7.0 Cz, Cg 6.4 7.0 CZ, cg VI1 4 4

VI11 3 7 6.5 7.2 c2 IX (3) k4 = 0 6 30 15 C1, C z , CE,

X. 5 4 3.8 4.0 C I

XI1 (3) k-4 = 0 5 3.3 5.0 Cz, C3 XIII 6 4 3.6 5.3 Accepted

x (3) k-g= 0 5 CS

XI (3) k6 = 0 6 6.6 7.1 cz

a Numbering refers to the vector diagrams that are represented in Figs. 3 and 6.

Rate equations:

Reduced equation

e N = 50 for models I and II; N = 136 for models 111 through XIII. Rejection criteria: C,, large sum of squares and standard error;

CZ, large relative uncertainty in parameters (>loo%); Car negative parameter; C4, term rejected by F test; C6, unacceptable visual fit; Cs, no convergence; C7, no model.

FIG. 4. Linearization of integrated rate Equation 1 for t- butylhydroperoxide at 2 mM constant GSH and various con- centrations of mercaptosuccinate. Each line was obtained by least squares linear regression of the points (each r P 0.99). From top to bottom, mercaptosuccinate concentrations are 18, 15, 12,9, 6, 3, and 0 +M, respectively.

of glutathione peroxidase is still consistent with Equation 2, with the apparent 41 unchanged (constant slope) and the apparent being a linear function of inhibitor concentration (intercept pattern). Since the ordimtes on Figs. 4 and 5 are

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Mechanism and Inhibition of Glutathione Peroxidase

I I - I80 7

e W - 0 03625 084 I 3 * E 40 55 70

Ln(Ao/A,-Pt ) /P f (rnM")

FIG. 5. Linearization of integrated rate Equation 1 for t- butylhydroperoxideat 3.8 p~ constant mercaptosuccinate and various GSH concentrations. Each line was obtained by least squares linear regression of the points (all r 0.985). From top to bottom, GSH concentrations are 0.75, 1.00, 1.50, and 2.50 mM, re- spectively. The line for 2.0 mM GSH was omitted for clarity.

proportional to Eo, the observation of parallel lines when Eo is fixed rules out noncompetitive types of inhibition (with respect to GSH). It also rules out interaction with E. On Fig. 5, the slope, the apparent &, is also independent of GSH concentration, whereas the apparent @2 decreases nonlinearly with GSH concentration.

Four simple mechanisms of inhibition were then consid- ered, and the corresponding King-Altman patterns were drawn for derivation of rate equations. These four patterns are shown in Fig. 6 as I11 through VI. They involve either the formation of a dead-end complex with an oxidized form of the enzyme or the appearance of an alternate pathway between two enzyme forms. E is the most reduced form of the enzyme, and it has the properties of a selenocysteine selenolate (5 , 15, 16). Although complete knowledge of the redox states of F and G is not required, for discussion purposes they may be assigned the properties of a selenenic acid, PSeOH, and of a thioselenenate, PSeSR, respectively.

Inspection of the four rate equations derived from models I11 to VI (Fig. 6) indicates that they are all compatible with both Equation 2 and a slope independent of inhibitor concen- tration. After nonlinear least squares fitting, models I11 to VI were rejected as shown in Table 111. The corresponding fits were poor at either constant GSH or constant inhibitor con- centration, and in model V a negative constant was obtained. These results suggested that a more complex function of GSH concentration was necessary to describe the observed inhibi- tion.

Other simple mechanisms can be postulated. These mech- anisms fall into two general categories: those implying the formation of a mixed disulfide RSSG and those involving an interaction of E with the inhibitor. The total amount of GSSG consumed for a given amount of hydroperoxide was independ- ent of inhibitor concentration, and the inhibition was not relieved during hydroperoxide consumption. Hence, the for- mation of a mixed disulfide of GSH is excluded because it would imply a very fast sulfhydryl-disulfide interchange with GSH, a process that would be strongly pH-dependent. This substantiates the conclusions based on the linear least squares fits to the integrated rate expression already discussed where interactions with E were also eliminated.

A number of more complex King-Altman patterns were accordingly explored, and the corresponding rate equations were assessed by nonlinear least squares fitting. The most significant models are represented as VI1 to XIII in Fig. 6. The results of the nonlinear least squares analysis of the derived equations are summarized in Table 111. Often equa- tions needed to be reduced to eliminate unconstrained degrees of freedom. These simplifications are analogous to grouping k2 and k3 in the uninhibited pattern.

I U E

G-F A i x 'v H

Y E

P I I E

E E

H

1047

X T E X I I E

FIG. 6. Kinetic models for inhibition of glutathione peroxi- dase by mercaptosuccinate. Each model is represented by a basic vector diagram from which King-Altman calculations can be done. E, F, and G are the enzyme forms described on Fig. 3 and are always connected as shown in model I; H is an enzyme-inhibitor complex; A, B, and I are the concentrations of hydroperoxide, GSH, and inhibitor, respectively; and k4, ks, k 4 , and k-5 are rate constants.

A comparison of these equations as descriptors of the data is summarized in Table 111. Clearly, none of the equations derived from models VI1 to XI was acceptable. In model X, the term in ( I ) / @ ) was systematically rejected (cf Table 111). An arbitrary deletion of this term led to Equation X, in Table 111. As shown in Table 111, this fit was statistically acceptable. All efforts to generate a viable model corresponding to this equation failed. The step responsible for the existence of an (I)/@) term was, therefore, arbitrarily modified by replacing k--4 with k-@) to yield model XIII. The corresponding equa- tion gave a good fit to the entire set of data, as detailed in Table 111. However, model XI11 imposes the new constraint that F should contain one molecule of bound GSH or be obtained from the decomposition of an intermediate which contained GSH. This constraint is incompatible with F having selenium in the form of a selenenic acid. As discussed below,

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1048 Mechanism and Inhibition of Glutathione Peroxidase

other mechanistic interpretations will yield models whose corresponding rate equations are kinetically equivalent to Equation XIII. The actual fit is shown in Fig. 7, where 14 curves (total of 136 points) were simultaneously adjusted to this equation by nonlinear regression. The highest relative uncertainty in the parameters involved was 17%, which is generally considered as acceptable (40). Since each set of absorbance curves was obtained with different solutions of enzyme and chemicals, a significant level of experimental fluctuations would be expected. For all of the previous models tested, k-, was replaced with k-4(B). The reduced equations usually resulted in a form that had been tried previously and gave unsatisfactory results for all equations.

Mechanistic Interpretation-Indirect information on the redox forms of the enzyme has been obtained in several ways. First, the most reduced form of the enzyme, E, apparently contains selenium in the form of a selenocysteine selenolate at physiological concentration of GSH (5, 15, 16). Second, x- ray diffraction patterns of glutathione peroxidase from bovine red blood cells support the complete equivalence of the 4 atoms of selenium/tetramer and rule out the possibility of intramolecular diselenide formation (41). Accordingly, start- ing from a selenocysteine selenolate, a two-electron reduction of a single molecule of hydroperoxide (kinetic constraint) to the corresponding alcohol would yield a (+II) oxidation state

30

20

10

g0 \

r - 20 3-

t

IO

0

HYDROPEROXIDE CONCENTRATION (pM)

rived from model XI11 to experimental data for inhibition of FIG. 7. Nonlinear least squares fitting of the equation de-

glutathione peroxidase by mercaptosuccinate. The lines repre- sent the theoretical function whereas points are experimental data obtained by analysis of entire progress curves at limiting concentra- tion of t-butylhydroperoxide. Three sets of data were simultaneously used for estimation of the parameters. A , data were obtained in the absence of inhibitor: from top to bottom, GSH concentrations are 2.0, 1.0,0.5, and 0.25 mM, respectively. €3, data were obtained at constant GSH concentration (2 mM): from top to bottom, mercaptosuccinate concentrations are 3, 6, 9, 12 , 15, and 18 PM. C , data were obtained at constant mercaptosuccinate concentration (3.8 PM): from top to bottom, GSH concentrations are 2.0, 1.5, 1.0, and 0.75 mM, respec- tively. The line for 2.5 mM GSH was omitted for clarity.

of selenium. This oxidized form, F, of glutathione peroxidase should be a selenenic acid (PSeOH). This would not preclude that the latter is kinetically unobserved due to very rapid transformation into another selenium (+II) derivative, e.g. a thioselenenate, PSeSR, or an ester. Indeed, if a sulfhydryl group, e.g. a cysteine residue or a noncovalently bound GSH, were present at the active site of E, it would be unlikely that a selenenic acid could be formed with a significant lifetime since this type of electrophile should react very rapidly with sulfhydryl groups. Instead, a pseudo ter-reactant system would directly yield a thioselenenate, F. This reasoning led to the two proposed mechanisms, a and b, in Fig. 8. However, these are not the only mechanisms consistent with the ob- served kinetics.

As further support of either interpretation, no trapping of a selenenic acid was observed with 20 mM of either cyanide, thiocyanate, hydrazine at pH 7.7, or dimedone at pH 9.0, all of which are used to trap protein sulfenic acids (42). On the other hand, the active site selenium can be slowly trapped by cyanide at alkaline pH with subsequent loss of selenium when the enzyme has been air-oxidized in the presence of GSH (43). This may indicate the hydrolytic cleavage of a thiosele- nenate and the trapping of the resulting selenenic acid, which is not normally observed in the catalytic cycle. The need for a sulfhydryl group in proximity to the selenium can be further rationalized as necessary to prevent overoxidation of the selenium, which would inactivate the enzyme. Additionally, one of the oxidized forms of glutathione peroxidase apparently contains one GSH bound at the active site (17). Amino acid analysis indicates the presence of one cysteine residue in glutathione peroxidase (15), which is required for model b to be feasible. The high specificity observed for the first reduc- tion step from F to G supports the existence of a specific recognition site for GSH although GSH binding could not be demonstrated with carrier-free (35S)GSH (17). Finally, one histidine residue has been identified at the active site of glutathione peroxidase (41); one common function of histidine is to modulate the protonation state of sulfhydryl groups.

For mechanism b to be valid, the inhibitor sulfhydryl must

/ 6SH

181 Ibl

FIG. 8. Two proposed mechanisms for glutathione peroxi- dase inhibition by mercaptosuccinate (RSH). The most reduced form of the enzyme has selenium in the form of a selenocysteine selenolate. Other redox forms of selenium correspond to thioselenen- ate intermediates. In mechanism a, . . . represents noncovalent bind- ing of GSH at the active site. In mechanism b, a cysteine residue in addition to a selenocysteine residue is involved at the active site. All reaction steps in either model can be visualized as nucleophilic displacements. These two models are kinetically equivalent to model XI11 in Fig. 6. In mechanism b, the very high specificity of the step that follows reduction of hydroperoxide may be due to the existence of a transient Michaelian complex with a very rapid reactional decomposition. This complex is not represented in the figure.

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Mechanism and Inhibition of Glutathione Peroxidase 1049

preferentially attack the sulfur and GSH must preferentially attack the selenium. This interpretation can be rationalized as a result of the high specificity for GSH in the first reduction step. Furthermore, if the inhibitor reacted with the selenium, the mechanism would no longer fit the observed kinetics. Steps determined to be kinetically irreversible would be re- versible as the same intermediate would be formed in two portions of the mechanism. These arguments cause some bias toward mechanism a, but this bias is insufficient to reject b.

Other interpretations of the kinetic data in this study would imply higher oxidation states for the selenium and covalently bound GSH. Although not disproved, these mechanisms are more complex because they would preclude the existence of a selenolate group within the catalytic cycle and imply the formation of selenium (+IV) intermediates within the enzyme cycle.

When the parameters obtained from the nonlinear least squares program are analyzed to obtain estimates of the rate constants as per model XIII, it is evident that there are more degrees of freedom than constraints. However, certain gen- eralities can be made. kl is 730,000 mM" min-l, with a relative uncertainty as estimated in the nonlinear least squares anal- ysis of 1.4%. The reduction steps, kz and k3, are both greater than 70,000 m"' min" as kz X k S / ( k z + k3) = 70,500 mM-' min-' with a relative uncertainty of 4.0%. Additionally, (k2 x k3 X k-4)/(kZ X K5 + k3 X k4 X k-, + ks X k-J = 195, with a relative uncertainty of 17%. The last constraint states that ( k , x k3 x k-.,)/(k, x k5) = 0.72, with a relative uncertainty of 9.4%. The following expressions are useful to examine the relative magnitudes of the rate constants: k5 = k3 X k4/[(0.0037 x k4) - k- , - kz] and k4 > 270 x ( k 2 + k-,). These constraints allow all rate constants in models a and b to have values that are positive and much less than the diffusion limit.

The reason most P-mercaptocarboxylic acids are able to compete so efficiently with GSH in trapping the oxidized forms of glutathione peroxidase is not clear. It is noteworthy that these compounds do not recycle the reduced form E of glutathione peroxidase by attack on sulfur and formation of a mixed disulfide of GSH but, instead, yield H. This indicates a strong preference for attack on selenium. Of great interest are the recent reports that mercaptocarboxylic acids are able to trap otherwise unstable Se(+II) and Te(+II) salts (44, 45). Hence, the possibility that they could also very rapidly trap organic Se(+II). This suggests that mercaptosuccinate could interact with other selenoproteins. The particularly strong inhibition observed with mercaptosuccinate may be due to the presence of two carboxylate groups. This could enhance competition with GSH for an electrostatic binding site or yield a transition state analog (46).

In conclusion, two significant results of this study are (i) the kinetic demonstration that a selenenic acid cannot be one of the three redox forms of the enzyme and (ii) the finding that mercaptocarboxylic acids are able to form unproductive complexes with the oxidized forms of glutathione peroxidase active site.

The potential applications of mercaptocarboxylic acid in- hibitors may eventually prove interesting. Mercaptosuccinate, dimercaptosuccinate, /3-mercaptovaline, and cr-mercaptopro- pionylglycine can be used in vitro specifically to block sele- nium-glutathione peroxidase activity. With high specificity, it may be possible to obtain new insights as to the function of selenium and/or glutathione peroxidase by using some of these inhibitors in viuo. Given its high specificity, mercapto- succinate may not affect other biological components to a significant extent. This is not to say that all P-mercaptocar- boxylic acids are devoid of known effects; for example, thio-

salicylate (2-mercaptobenzoate) serves as a potent inhibitor of respiratory chains through uncoupling of oxidative phos- phorylation (47).

It is interesting to note that three of the most potent inhibitors found in this study, namely, thiomalate (mercap- tosuccinate), penicillamine (P-mercaptovaline), and cr-mer- captopropionylglycine, are currently used as slow-acting drugs in the treatment of rheumatoid arthritis. Whereas thiomalate has mostly been used as a complexing agent for gold (in the drug Myochrysine), recent studies indicate that thiomalate may be an active part of aurothiomalate (48, 49). The mech- anism of action of such drugs is unknown. However, intracel- lular GSH concentration was shown to increase in the eryth- rocytes of a patient treated with penicillamine (48). It is generally accepted that the glutathione peroxidase pathway accounts for a major part of GSH consumption (50). Given the apparent importance of neutrophils and macrophages in this inflammatory disease, it seems relevant that (i) GSSG has recently been shown to be an essential activator of leu- kocyte collagenase (51) and (ii) GSH is a limiting substrate in the biosynthetic pathway to leukotrienes (52). These find- ings justify further attempts to assess the possibility that Se and/or glutathione peroxidase is involved in the etiology or evolution of rheumatoid arthritis.

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J Chaudiere, E C Wilhelmsen and A L Tappelmercaptocarboxylic acids and other mercaptans.

Mechanism of selenium-glutathione peroxidase and its inhibition by

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