the journai. of biological chemistry november 25, pp. … · the journai. of biological chemistry...

THE JOURNAI. OF BIOLOGICAL CHEMISTRY Vol. 253, No. 22, Issue of November 25, pp. 8109-8119, 197R Prmled in [J.S.A.

Active Site Cysteinyl and Arginyl Residues of Rhodanese A NOVEL FORMATION OF DISULFIDE BONDS IN THE ACTIVE SITE PROMOTED BY PHENYLGLYOXAL*

(Received for publication, April 20, 1978)

Litai Weng,$ Robert L. Heinrikson,@ and John Westley

From the Department of Biochemistry, University of Chicago, Chicago, Illinois 60637

Chemical modification studies of bovine liver rhodanese have underscored important distinctions between free rhodanese and the catalytic intermediate in which the sulfane atom of the sulfur donor is bound covalently to the enzyme (sulfur-rhodanese). Treat- ment of free rhodanese with near-stoichiometric quantities of either iodoacetate or phenylglyoxal results in the rapid modification of the essential sulfhydryl group of Cys-247 and the consequent inactivation of the enzyme. Analysis of rate data for the iodoacetate reaction showed that the apparent pK of this group is 7.8 in free rhodanese and 6.7 to 7.0 in complexes of the enzyme with analogs of sulfur donor substrates, in agreement with the previous inference from steady state kinetic observations. Inactivation of free rhodanese by phenylglyoxal in the presence of cyanide was shown to be caused by a novel reaction in which disulfide bonds are formed between Cys-247 and either Cys-254 or Cys-263. In contrast to these results with free rhodanese, the sulfur-substituted enzyme is not inactivated by iodoacetate and is only relatively slowly inactivated by treatment with substantial molar excesses of phenylglyoxal. The loss of enzyme activity in sulfur-rhodanese does not involve cysteinyl residues but can be correlated with the modification of guanidino groups, notably that of Arg-186, the side chain of which may play a role in substrate binding. These chemical modification studies have implications with respect to the chemical mechanism of rhodanese catalysis and the interpretation of the x-ray crystallographic analysis of this enzyme.

Rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) catalyzes the transfer of a sulfane sulfur atom from an anionic donor substrate to a thiophilic acceptor by a mechanism involving a stable covalent sulfur-substituted enzyme intermediate (l-3), hereafter referred to as sulfur-rhodanese. This form of the enzyme is to be distinguished from that designated free rhodanese in which the covalently bound sulfur has been removed by the addition of cyanide. Since free rhodanese is quite unstable, the preparative procedures com- monly employed for isolation of the enzyme (4, 5) include

* This research was supported by Grants GB-29098, BMS 74-18144, BMS 75-03147, and BMS 75-23506 from the National Science Foun- dation and by Grant PHS GM-18939 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Some of this work was performed in partial fulfillment of the requirements for the Ph.D. degree at the University of Chicago. Present address, Department of Pharmacology, Stanford University, Stanford, Calif. 94305.

5 To whom all correspondence should be addressed.

thiosulfate at each step, and the final crystalline protein product is sulfur-rhodanese. The x-ray crystallographic analysis of the enzyme has been performed on sulfur-rhodanese; attempts to crystallize free rhodanese have been unsuccessful to date.

The two preceding papers in this series (6, 7) provided details concerning the covalent structural analysis of rhodanese. The enzyme is a single polypeptide containing 293 amino acid residues. Information concerning the active site of rhodanese has been obtained both by solution chemistry and by x-ray crystallography. Rhodanese contains 4 cysteine residues at positions 63, 247, 254, and 263 in the polypeptide (6, 7). There are no disulfide bonds in the molecule. The sulfhy- dry1 group of Cys-247 has been identified as the site of covalent attachment of the sulfane atom in sulfur-rhodanese (8). Confirmation of this assignment has been made by x-ray crystallography of sulfur-rhodanese at 2.5 A resolution in which the persulfide linkage with S” of Cys-247 was clearly documented (9, 10). Steady state kinetic studies have shown that the apparent pK of the enzymic nucleophile involved in cleavage of the sulfur-sulfur bond of the sulfur-donor substrate is approximately 6.5 (11).

Other features of the active center that have been predicted from chemical studies include a cationic binding site (12) and a hydrophobic site (13). Crystallographic analysis has shown that the side chains of Phe-212, Phe-106, Tyr-107, Trp-35, and Val-251 provide a hydrophobic wall of the active site and that the guanidino and e-NH2 groups of Arg-186 and Lys-249, respectively, are positioned in such a way as to suggest possible participation in substrate binding (9, 10). The implication of amino groups in rhodanese catalysis was suggested by inactivation studies with pyridoxal5’-phosphate, although no specific sites of modification were identified (14).

The studies described in this communication were designed to characterize the pH dependency and specificity of the alkylation of Cys-247 by iodoacetate and to explore the possible catalytic involvement of arginyl residues in the rhodanese mechanism. During the course of this work, a novel reaction of phenylglyoxal was observed in the cyanide-dependent promotion of disulfide bond formation in the active center of rhodanese. These findings demonstrate clear differences between the reactivities of active site residues in free rhodanese and sulfur-rhodanese and are of importance rela- tive to the x-ray crystallographic interpretation of enzyme conformational transitions during catalysis.

EXPERIMENTAL PROCEDURES

Some materials and experimental procedures were as reported in the preceding papers (6, 7) with the following additions and excep- tions.

Materials

The following chemicals were obtained from Aldrich: phenylglyoxal monohydrate, methylglyoxal (40% aqueous solution), glyoxal (40%

8109

by guest on April 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8110 The Active Site of Rhodanese

aqueous solution), benzil, benzoin, benzaldehyde, phenylacetaldehyde, 2,3-butanedione, 1,2-cyclohexanedione, and glycolaldehyde. 2- Hydroxyacetophenone, I-phenyl-1,2-propanedione, benzenesulfonate sodium salt, and butanesulfonate sodium salt were purchased from Eastman. Acetophenone was obtained from Fisher. [2-‘4C]Phenyl- glyoxal monohydrate and [‘4C]potassium cyanide were purchased from New England Nuclear. Benzoylformoin (1,2,4-butanetrione-3- hydroxy-1,4-diphenyl) was prepared by the method of Karrer and Segesser (15) and of Ruggli et al. (16). The nuclear magnetic reso- nance spectrum of this compound in CD:,OD solution showed 3 protons (multiplet) at S 7.35, 5 protons (multiplet) at 6 7.56, and 2 protons (multiplet) at S 8.27.

Methods

Assay of Enzymic Activity-Rhodanese activity was measured by the rate of thiocyanate formation from thiosulfate and cyanide (17, 18). The assay mixture contained 50 pmol of sodium thiosulfate, 50 pmol of potassium cyanide, and 40 prnol of potassium dihydrogen phosphate in a final volume of 1.0 ml at 25°C. The reaction was initiated by adding microliter quantities of enzyme and stopped by adding 0.5 ml of 15% formaldehvde. The amount of thiocvanat,e formed was then measured as absorbance at 460 nm after addition of 1.5 ml of a dilute ferric nitrate reagent made by adding 0.5 volume of deionized water to the reagent specified by Sorbo (17).

Determination of Protein Concentration-Protein concentrations of solutions of rhodanese were determined spectrophotometrically at 280 nm, with an absorptivity coefficient of 1.75 cm’/mg (17), or by amino acid analysis.

Determination of Sulfhydryl Groups-The sulthydryl groups of native and modified rhodanese were determined according to the method of Ellman (19) in 2% (w/v) sodium dodecyl sulfate, using 12,500 as the molar extinction coefficient of reduced 5,5’-dithiobis(2- nitrobenzoic acid) in 2% sodium dodecyl sulfate at 412 nm (19).

Inactivation of Free Rhodanese with [2-‘4CJZodoacetate-To an 18-mg sample (0.55 pmol) of rhodanese in 2.8 ml of 0.1 M glycine buffer, pH 9.0, was added 10 ~1 of 0.25 M KCN to remove enzyme- bound sulfur (20), followed by 1~1 of 0.75 M [2-“Cliodoacetic acid (1.4 x 10” dpm/mol) in 1 N NaOH. The reaction was allowed to proceed at 25°C and l-y1 aliquots were withdrawn at suitable intervals and assayed for enzymic activity. When the enzymic activity had decreased to 2.5% of the zero time value (about 42 min), the reaction was stopped by the addition of 15 ~1 of 2-mercaptoethanol, and stirring was continued for 2 h. The reaction mixture was then dialyzed exhaustively against water and lyophilized.

Isolation of Labeled Peptide-A small portion of the lyophilized, partially S-[‘4C]carboxymethylated rhodanese was hydrolyzed for amino acid analysis. The rest was reduced and completely carboxymethylated with nonlabeled iodoacetate and then citraconylated by procedures described in the preceding papers (6, 7). After desalting and lyophilization, the citraconylated carboxymethylcysteinylrho- danese derivative was hydrolyzed with 2% by weight of trypsin in 2 ml of 0.2 M N-ethylmorpholine acetate, pH 8.2, for 3.5 h at 37°C. The digestion mixture was then passed through a column (2.5 x 180 cm) of Sephadex G-50 in 0.1 M NH4HCOs. Fractions containing radioactivity were compared to those known from earlier sequence analysis to contain cysteinyl peptides.

Treatment of Sulfur-Rhodanese with [2-‘4CJZodoacetic Acid-Fifteen milligrams (0.45 pmol) of crystalline sulfur-rhodanese was dissolved in 1.5 ml of 0.1 M glycine buffer, pH 9.0, containing 0.005 M Na2S203. Ten microliters of 1.06 M [2-‘4C]iodoacetic acid (120,090 dpm/,rrmol) in 1 N NaOH was added to the enzyme solution at room temperature. At suitable intervals, l-p1 aliquots were withdrawn and assayed for enzymic activity. After 75 min, the reaction was stopped by adding 0.2 ml of 2-mercaptoethanol, and stirring was continued for 2 h. The reaction mixture was then dialyzed and lyophilized. A small portion of this lyophilized rhodanese was analyzed for amino acid content and radioactivity.

Alkylation Reactions of Free Rhodanese at Different pH Values-Reaction mixtures containing approximately 2.8 X IO-” M

rhodanese, 8 X lo-“ M KCN, and 3 X 10e4 M disodium EDTA were buffered with phosphate over the pH range 6.0 to 10.0, ionic strength 0.1 at 25°C. The phosphate buffers at pH values 6.0, 6.4, 6.8, 7.2, and 7.6 were prepared from potassium dihydrogen phosphate and disodium hydrogen phosphate (21). Buffers at pH values of 8.0, 8.5, and 9.0 were prepared from potassium dihydrogen phosphate and Tris and those at 9.5 and 10.0 from potassium dihydrogen phosphate and ethanolamine. [2-‘4C]Iodoacetate (1.2 x lo” dpm/pmol) was added to the reaction mixture at a final concentration of approximately 2.4 x

IO-” M. At suitable intervals, l+l aliquots were withdrawn and assayed for enzymic activity until the activity had decreased to less than 5% of the zero time value. After inactivation, the reaction mixture was dialyzed exhaustively against water, lyophilized, and then subjected to procedures for amino acid analysis and measurement of radioactivity.

Determination of Rate Constants for Alkylation Reactions of Rhodanese-Reaction kinetics of the alkylation of rhodanese were followed by the rate of inactivation of the enzyme. A Hewlett-Packard 2000 C digital computer with a BASIC program providing a best fit line by least squares linear regression was used to calculate the rate constants according to the general rate equation:

In 2 = kt (1)

where A is the enzymic activity and k is the rate constant. Determination of the pK’ Value of the Group Alkylated in the

Active Site of Rhodanese-The logarithm of the apparent second order rate constant (kohr) for the alkylation reactions was plotted against pH. Plots of this form are composed of linear portions having integral slopes connected by smooth curves. The pK’ values are determined from the points of intersection of the extrapolated linear portions (22).

Theoretical curves for particular pK values were obtained from computer-simulation studies by direct programming of the equation (23):

kohs = kz

1 + H+/K, (2)

where koba is the rate constant obtained from the measured rate and k2 is the rate constant for the ionic species of the acid HA whose ionization constant is K,.

For studies done in the presence of an inhibitor (I),

kuhh = kz

H+ K,

l*z I+[11 ( 1

(3)

where K, is the inhibitor constant. Reaction of Phenylglyoxal with Sulfur-Rhodanese-Six milli-

grams (0.18 pmol) of crystalline sulfur-rhodanese was dissolved in 1 ml of phosphate buffer at pH 7.8, ionic strength 0.1. A solution of phenylglyoxal (12 pmol) in 100 ~1 of water was added to the enzyme solution and reaction was carried out at 25°C. At suitable intervals, 1-d samples were removed for assay of enzymic activity. When the activity had decreased to 8% of the zero time value (about 60 min), a small portion of the solution was dialyzed against several changes of 5% acetic acid for 1 day, lyophilized, and then hydrolyzed for amino acid analysis. The rest of the protein was freed from reagent by passage of the reaction mixture through a column (2.5 x 35 cm) of Sephadex G-75, equilibrated at 4°C with phosphate buffer, pH 7.8, ionic strength 0.1. The resulting protein fractions were concentrated to about 4.5 mg/ml. The sulfhydryl content of this phenylglyoxal- inactivated sulfur-rhodanese was determined with Ellman reagent.

When the reaction between phenylglyoxal and sulfur-rhodanese was carried out under the conditions described above for 3 h, the activity remained constant after 1.5 h at a level about 4% of the zero time value, i.e. the phenylglyoxal-inactivated sulfur-rhodanese had some residual enzymic activity. The kinetics of the thiosulfate-cyanide reaction catalyzed by this rhodanese derivative was studied as described below.

For identification of the active site residues modified, a difference labeling technique was employed. Sulfur-rhodanese (130 mg, 4 pmol) was first reacted at 25°C with 240 pmol of phenylglyoxal in the presence of 0.17 M Na$SzO:3 in 18 ml of phosphate buffer, pH 7.8, ionic strength 0.1. Enzyme activity was measured at various times as described above. After 50 min, a small portion of the solution was dialyzed prior to acid hydrolysis and amino acid analysis. The rest of the protein was freed from reagents by passage of the reaction mixture through a column (3.2 x 45 cm) of Sephadex G-75, equilibrated at 4°C with phosphate buffer, pH 7.8, ionic strength 0.1, containing 0.005 M Na&03. The desalted protein fractions were concentrated to 14 ml (about 8.5 mg/ml). Approximately a 50.fold molar excess (180 pmol) of [2-“‘C]phenylglyoxal (10” dpm/Zrmol) was added and the reaction was carried out at 25”C, now in the absence of a large excess of substrate. When the enzymic activity had decreased to 15% of the zero time value (about 33 min), the reaction mixture was dialyzed


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

The Active Site of Rhodanese 8111

exhaustively against 5% acetic acid and then lyophilized. Kinetic Studies of Phenylglyoxal-inactivated Sulfur-Rho-

danese--Steady state kinetics of the thiosulfate-cyanide sulfur transfer reaction catalyzed by the phenylglyoxal-inactivated sulfur-rhodanese was followed by measuring thiocyanate formation under the standard assay conditions except that thiosulfate concentration was varied from 0.05 to 0.5 M. As a control, kinetic analysis of the same reaction of fully active rhodanese was carried out under the same conditions except that thiosulfate concentrations were varied from 0.0015 to 0.05 M. The initial velocity data expressed as molar thiocyanate formed per s per molar enzyme were plotted in double reciprocal form and also processed with a Hewlett-Packard 2000C digital computer using a BASIC program for least squares fitting to a hyperbolic function. From this fit, the kinetic constants K,n and V,,,,/Eo and their standard error values were obtained. These constants could be related directly to K, and h+z since it was previously shown that K, for thiosulfate approximates a true equilibrium constant and that V is dominated by h+.~ (24).

Identification of the Residues Labeled by [‘4CJPhenylglyoxal in Sulfur-Rhodanese-The [‘4C]phenylglyoxal-inactivated sulfur-rhodanese (about 2.5 pmol) was reduced, carboxymethylated, and then citraconylated under the usual conditions except that reduction was carried out at room temperature for only 15 min. The solution of this rhodanese derivative (72 mg) was desalted by passage of the reaction mixture through a column (2.5 x 50 cm) of Sephadex G-25 in 0.2 M N- ethylmorpholine acetate, pH 8.0, at 4°C and then concentrated to approximately 7 ml. Aliquots of this protein solution were removed for amino acid analysis and measurement of radioactivity. The rest of the protein solution was subjected to digestion with 3% by weight of trypsin for 3.5 h at room temperature. The digestion mixtures were separated by passage through a column (2.5 x 180 cm) of Sephadex G-50 in 0.1 M NH4HC03. The fractions containing radioactivity were combined, lyophilized, and then subjected to further analyses to identify the labeled residues.

Reaction of Free Rhodanese with Phenylglyoxal and Some Other Carbonyl Compounds in the Presence of Cyanide-Crystalline rhodanese was dissolved in phosphate buffer, ionic strength 0.1, pH 7.8, at protein concentrations of 5 to 10 mg/ml (1.5 X 10m4 to 3.03 X 1O-4 M); a nearly 3-fold molar excess of KCN and a nearly equimolar concentration of disodium EDTA were added, followed by a slight molar excess of phenylglyoxal monohydrate. The reaction mixture was kept at 25”C, and l-p1 aliquots were removed at intervals for assay of enzymic activity until less than 1% of the activity remained. After inactivation, an aliquot of the enzyme solution was reactivated by prolonged treatment with 1 M Na&O:s at 0°C. The rest of the reaction mixture was passed through a column of Sephadex G-75 equilibrated at 4°C with phosphate buffer, pH 7.8, ionic strength 0.1, or 0.1 M NH~HCOZ, to determine the molecular weight of the inactivated enzyme and to separate small molecular reactants and products from enzyme.

In one experiment with “C-labeled phenylglyoxal, the labeled reagent was used after dilution with nonlabeled reagent to specific radioactivity 340,090 dpm/amol. In another experiment, where only 50% inactivation was sought, a 0.5 molar ratio of the reagent to enzyme was used.

Reactions with methylglyoxal, glyoxal, benzil, benzoin, benzaldehyde, acetophenone, 2-hydroxyacetophenone, phenylacetaldehyde, 2,3-butanedione, 1-phenyl-l$propanedione, 1,2cyclohexanedione, glycoaldehyde, and benzoylformoin were carried out in a similar manner. Some of these reagents are not soluble in water; it was therefore necessary to prepare them in methanol and to add aliquots of the methanol solutions to the reaction mixtures; the same amount of methanol was added to the control samples. In cases in which no significant inactivation occurred with a IO-fold molar excess of reagent, the observations were stopped at 2 h.

Tryptic Digest of Phenylglyoxal-inactivated Free Rhoda- nese-After passage through a Sephadex G-75 column, phenylglyoxal- inactivated free rhodanese was concentrated by ultrafiltration with an Amicon ultrafilter (LJM-10 membrane) to about 10 mg/ml. The concentrated protein (1.8 pmol) was then subjected to carboxymethylation without prior reduction by adding the protein solution to 10 ml of 8 M guanidine HCl solution, pH 8.6 (8 M guanidine HCl, 0.4 M N- ethylmorpholine acetate, 0.045 M EDTA) containing 500 pmol of iodoacetate, where it was allowed to react for 20 min in the dark. In another experiment, designed to minimize disulfide exchange under basic conditions (251, phenylglyoxal-inactivated free rhodanese was added directly to a solution of 8 M guanidine HCl containing iodoacetate without prior gel filtration. The carboxymethylated rhodanese

was citraconylated, desalted, and lyophilized, followed by tryptic digestion with 2% trypsin in 4 ml of 0.2 M N-ethylmorpholine acetate, pH 8.2, for 3.5 h. Separation of tryptic peptides was made by gel filtration on a column (2.5 x 190 cm) of Sephadex G-50 in 0.1 M NH4HC03. Fraction I from this separation (cf Fig. 8) was reduced and [Wlcarboxymethylated as previously (261, except that 15 al of 2-mercaptoethanol was used and the specific radioactivity of the [‘4C]iodoacetate was 550,000 dpm/pmol. The peptides thus obtained were separated on the same column of Sephadex G-50 described above.

RESULTS

Identification of the Cysteine Residue Involved in the Inactivation of Free Rhodanese with [2-‘4C]Iodo- acetate-Free rhodanese is completely inactivated upon treatment with only a 1.4-fold molar excess of [2-“‘Cliodoacetate, based upon a protein molecular weight of 33,000. Amino acid analysis of the inactivated enzyme showed only 1 residue of carboxymethylcysteine per mol of rhodanese. The specific radioactivity of this rhodanese derivative was identical with that of the reagent (approximately 1.4 X 10” dpm/pmol), indicating again that only 1 cysteine residue had been ‘%- alkylated. It was known from our sequence analysis of the tryptic peptides (Tc fragments) of citraconylated, carboxy- methylcysteinyl-rhodanese that the three cysteine-containing peptides can be completely separated from each other by gel filtration on a column of Sephadex G-50. The nomenclature for these peptides and the methods for their separation are described in the first paper of this series (6). Fraction II (Fig. 1 (6)) contained the peptide with Cys-247 (Tc17; Ala-230 to Arg-248); Fraction IV corresponds to the peptide with 2 cysteines, Cys-254 and Cys-263 (Tc18; Lys-249 to Arg-281); and Fraction V contains the peptide with Cys-63 (Tc7a; His- 51 to Arg-64). As noted before (6) in some digests the Arg- Asp bond is not cleaved by trypsin; then Cys-63 is contained in Fraction I (Tc7; His-51 to Arg-110).

In this experiment, the rhodanese derivative inactivated by reaction with [2-‘%]iodoacetate was reduced, carboxymethylated with nonlabeled iodoacetate, and citraconylated. The resulting rhodanese derivative was hydrolyzed with trypsin and the digest was passed through a column of Sephadex G- 50 (Fig. 1). More than 90% of the radioactivity emerged in a single peak (Peak II) in which was included the peptide containing Cys-247; the remaining few per cent of radioactivity were scattered in Peaks IV and V, which contained Cys-

1000

I 5

008 800 p

$ ;

t 006 600 -i

8

8 ;

5 004 400 5

E 5: 9 0.02 200

400 600 800 1000 1200

EFFLUENT VOLUME lml)

FIG. 1. Separation of tryptic peptides from citraconylated carbox- ymethylcysteinyl-rhodanese on Sephadex G-50 (superfine). Column, 2.5 x 190 cm; eluant, 0.1 M NH,HCO:%; flow rate, 30 ml/h. The native enzyme (0.45 pmol) was inactivated with a 1.4-fold molar excess of [2- “C]iodoacetate, then reduced and fully S-carboxymethylated with nonlabeled reagent. Fractions of 6 ml were collected; O.l-ml aliquots of each fraction were measured for radioactivity.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


254, 263, and 63. Therefore, Cys-24’7 is, by far, the predomi- nant site of alkylation upon inactivation of free rhodanese with [“Cliodoacetate. These findings together with those published earlier (8) are consistent with the view that Cys- 247 is the active site cysteine in rhodanese.

In contrast to the result with free rhodanese, treatment of sulfur-rhodanese with a ZO-fold molar excess of [2-‘4C]iodoacetate resulted in no inactivation over a 75min period. Both amino acid analysis and radioactivity measurement of this rhodanese derivative showed that on the average, a total of only one-half of 1 cysteine residue had been “‘Calkylated per enzyme molecule. No further identification of the partially labeled cysteine residues was carried out. Presumably Cys- 247 was totally protected, while Cys-63, 254, and 263 could be labeled to some extent without affecting the enzymic activity.

pK’ Value of Cys-247 in the Presence and Absence of Substrate Analogs-In order to determine the pK’ value of the active site cysteine, the pH-dependency of the iodoacetate reaction was determined. The reaction between free rhodanese and a g-fold excess of iodoacetate followed pseudo-fast order reaction kinetics under all conditions of pH in the range between 6 and 10. Pseudo-first order rate constants were determined by plotting the logarithm of enzymic activity against time and determining the best fitting slope by computer calculation. These were converted to second order constants by division by the iodoacetate concentration. Amino acid analysis of rhodanese inactivated in this way gave 1.0 to 1.5 mol of S-carboxymethylcysteine/mol of enzyme; no other amino acids were modified. Radioactive measurement also indicated the incorporation of 1.0 to 1.5 mol of label/m01 of enzyme. All the 14C was accounted for in S-carboxymethyl-

1ooc

tot

2

1C

1.C

)-

)- 6

1 I I I

7 8 9 10

PH FIG. 2. pH-rate profiies for the reaction of iodoacetate with free

rhodanese in the absence (A) and presence (B) of benzenesulfonate.

20 40 60 80 SO-----i80

TIME (min) FIG. 3. Inactivation of sulfur-rhodanese by phenylglyoxal at pH

7.8 and 25°C. [E-S] = 1.8 X 10m4 M; [phenylglyoxal] = 1.2 x IO-” M.

cysteine. Although under some conditions, more than 1 cysteine was alkylated during inactivation, the pseudo-first order kinetics of inactivation indicated that only 1 cysteine is important for enzymic activity, in agreement with the conclu- sions derived above.

A plot of Kobs (logarithmic scale) uersus pH for the reaction of free rhodanese with iodoacetate is shown in Fig. 2 together with the results obtained from the reaction in the presence of 0.5 M benzenesulfonate. The pK’ values were determined graphically as 7.8 for free rhodanese and 6.75 for rhodanese in the presence of benzenesulfonate. The value calculated for the reaction in the presence of 0.5 M butanesulfonate was 7.05. The curve in Fig. 2 for the reaction of free rhodanese was calculated from Equation 2 with KS = 1.3 x 10’ Me’ min-‘, pK’ = 7.8. The curve for the reaction in the presence of benzenesulfonate was calculated from Equation 3 with KZ = 3.7 x 10’ M-’ min-‘, pK’ = 6.75, [I] = 0.5 M, and K, = 1.4 X lo-* M.

Inactivation of Sulfur-Rhodanese by Phenylglyoxal and Characterization of the Protein Derivative-When sulfur- rhodanese was treated with a 66-fold molar excess of phenylglyoxal to protein at pH 7.8 and 25”C, the enzyme was inactivated as shown in Fig. 3. This reaction followed pseudo- first order kinetics until 96% of the enzyme had been inactivated; the second order rate constant was 4.2 M-’ min-‘. No further loss of activity was observed during prolonged incubation for times up to 180 min. When the same experiment was performed in the presence of 0.17 M Na&Oz virtually no inactivation was observed over 50 min.

When the phenylglyoxal-inactivated rhodanese derivative was passed through a column of Sephadex G-75, the protein was eluted at the same position as sulfur-rhodanese, showing that no significant amounts of polymerized forms were present. The ultraviolet absorption spectrum of this rhodanese derivative showed higher absorp-cion at 250 nm compared to sulfur-rhodanese, indicating that phenylglyoxal was bound covalently to the enzyme.

Double reciprocal plots derived from steady state kinetic studies of the thiosulfate-cyanide reaction catalyzed by native rhodanese and by phenylglyoxal-inactivated enzyme with 4% residual activity are shown in Fig. 4. The kinetic constants K,,, and Vm,,/Eo for native rhodanese are 3.85 + 0.35 mM and 410 f 10 s-l, respectively. Corresponding values for the modified rhodanese are 553 r~_ 69 mM and 130 f 10 s-‘. These data show the kinetic effects of modifying rhodanese with phenylglyoxal:


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


10 A .

I 1 I

2 4 6 8

IO-*/[S,O,=] (M-‘)

I I I

5 10 15 20

[s,o,=]-’ CM-‘) FIG. 4. Kinetics of the rhodanese-catalyzed reaction of thiosulfate

with 0.05 M cyanide. A, thiosulfate concentrations were varied from 0.0015 to 0.05 M; B, thiosulfate concentrations were varied from 0.05 to 0.5 M.

K ‘So? was increased 140-fold after modification, while V,,,,,/ Efwas decreased 3-fold.

Amino acid analysis of a rhodanese derivative inactivated to the extent of about 92% by phenylglyoxal showed that 3.5 of the 20 arginyl residues had been modified. There was no change in the values for the other amino acids, within the limit of experimental error. Reaction with Ellman reagent showed that sulfhydryl groups had not been modified. This evidence indicates that phenylglyoxal inactivates sulfur-rhodanese by reaction with arginyl residues. The decrease in activity showed pseudo-fist order decay, raising the possibility that only one of the modified arginines directly affects the activity.

In an attempt to incorporate “C-labeled substituent more specifically into the active site region, sulfur-rhodanese was reacted first with phenylglyoxal in the presence of thiosulfate, and then with 14C-labeled phenylglyoxal following removal of substrate. In the fist phase of the experiment, the reaction of nonlabeled phenylglyoxal with 130 mg (4 pmol) of sulfur- rhodanese was carried out in the presence of 0.17 M Na2S203. An average of about 1.5 residues of arginine were modified without affecting the enzyme activity. After removal of the Na&03 substrate by gel filtration, the protein was treated with a 50-fold molar excess of [2-‘4C]phenylglyoxal. The resulting inactivation of the enzyme was accompanied by a further loss of about 3 arginine residues as shown by amino acid analysis and about 2.3 residues as shown by measurement of radioactivity. In the latter calculation, it was assumed that 2 phenylglyoxal molecules are bound per guanidino group of

arginine (27). Altogether, approximately 4.5 arginyl residues were modified, of which about 2.3 were with 14C substituent.

The rhodanese inactivated by phenylglyoxal in this difference-labeling experiment was reduced, carboxymethylated and citraconylated, and then desalted and concentrated to approximately 7 ml in 0.2 M N-ethylmorpholine acetate, pH 8. Amino acid analysis showed 17 arginine residues, compared to a value of 15.5 determined prior to this treatment. This analysis correlated well with radioactivity measurements which showed that approximately 1.5 arginine residues were “C-labeled following these procedures, as compared to 2.3 residues before. That is, under these basic conditions, 1.5 arginine residues were regenerated, of which 0.8 residue was 14C-labeled.

After tryptic digestion of this rhodanese derivative, the peptides were separated on a column (2.5 X 180 cm) of Sephadex G-50 in 0.1 M NH4HC03. The elution profile shown in Fig. 5 is similar to that in Fig. 1 except that the peaks are somewhat broader. Three major radioactive peaks, labeled I, III, and IV in Fig. 5 were detected. The rest of the radioactivity was distributed evenly in the remaining fractions, indicating nonspecific labeling of arginine. Fractions I through V were lyophilized for further study.

Identification ofArg-186 as the Major Site ofModification during Inactivation of Sulfur-Rhodanese by Phenyl- glyoxal-One-fourth of Fraction IV (Fig. 5) was decitraconylated in 5% HCOOH, then subjected to automated Edman degradation for 10 cycles. Five peptides were detected, as shown in Scheme 1 below:

1. Ala-Gln-Gly-m-Tyr-Leu-Gly-Thr-Gln-Pro-, ( -250 nmol)

183 186

2. Lys-Gly-Val-Thr-Ala-Cys-His-Ile-Ala-Leu-. (-250 nmol) 249

3. Ala-Leu-Val-Ser-Thr-Lys-Trp-Leu-Ala-Glu-. (-50 nmol) 8

4. Asn-Trp-Leu-Lys-Glu-Gly-His-Pro-Val-Thr-. (-50 nmol) 132

5. Gln-Val-Leu-Tyr-Arg-Ala-Leu-Val-Ser-Thr-. (-50 nmol) 3

SCHEME 1

In control tryptic digests, Fraction IV consists predomi- nantly of the second peptide in Scheme 1 (TcM), plus a small amount of the third and fourth peptides. The first peptide in Scheme 1, corresponds to two fragments, Tc14 and Tc15,

05L ~2000

E 04- -1500

ii

p

i

t 03. 3. - E 1000 51

5 oz- ; 0”

E a - 500 4” O.l-

400 600 800 1000 1200

EFFLUENT VOLUME (ml)

FIG. 5. Separation of tryptic peptides from S-carboxymethylated, citraconylated, [‘%]phenylglyoxal-inactivated sulfur-rhodanese (2.5 pmol) on a column (2.5 x 185 cm) of Sephadex G-50 (superfine) eluted with 0.1 M NH4HC03 (flow rate, 30 ml/h). Fractions of 6 ml were collected; 100~,al aliquots were assayed for radioactivity.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


which are present as one because trypsin failed to cleave at Arg-186. The fifth peptide, present in low yield in this fraction, was a combination of Tel and Tc2 in which Arg-7 was not cleaved.

Measurements performed on each cycle from automated Edman degradation showed the highest radioactivity in Cycle 4, consistent with the 14C-labeling of Arg-186. The radioactivity in Cycle 5 was 35% of that observed at Cycle 4. Whether this was due to overlap from Cycle 4 or labeling of Arg-7 is not clear. It should be noted that the radioactivity recovered was in the organic phase, since the reaction product of arginine with phenylglyoxal, unlike arginine itself, is extracted by organic solvents.

Another one-third of Fraction IV was decitraconylated and passed through a column (2.5 X 95 cm) of Sephadex G-25 in 5% HCOOH. Two fractions, detected by measurement of radioactivity and absorbance at 280 nm, respectively, were partially resolved. The amino acid analysis of fractions pooled from the leading edge of the first peak was that expected for the first peptide in Scheme 1, that is, the peptide from Ala- 183 to Arg-205 (Tc14 and Tc15). This result lends further support to the contention that Arg-186 is the site of modification by [‘4C]phenylglyoxal. Radioactivity measurements showed that approximately 50% of this arginine was 14C-labeled. None of the expected (6) Tc15 was detected by automated Edman degradation of Fraction V, so it was clear that Arg-186 had reacted nearly to completion. Therefore, despite the fact that only 50% labeling of Arg-186 was demonstrated directly, it is fair to assume that about 80 to 90% of this arginine was 14C-labeled originally and that the conditions of slightly alkaline pH employed during the workup of peptides led to loss of about 40% of the label (27).

danese by phenylglyoxal, methylglyoxal, and benzoylformoin are shown in Fig. 6. Rhodanese is also inactivated by glyoxal, but at a much slower rate. A great variety of additional carbonyl compounds was tested (CL “Experimental Proce- dures”) but unless they contained a-ketoaldehyde groups they did not inactivate, or react with, free rhodanese. The only exception observed thus far is benzoylformoin (Fig. 6); benzil does not inactivate the enzyme. All of these reactions were carried out in the presence of 3-fold molar excesses of cyanide added to strip the sulfur from sulfur-rhodanese. It was later discovered that the cyanide dependency of the inactivation of free rhodanese by phenylglyoxal is absolute. No loss of enzyme activity is observed when cyanide is removed by gel filtration from free rhodanese prior to the addition of phenylglyoxal, but inactivation takes place at the usual rate upon addition of cyanide to the reaction mixture. The role of cyanide in the reaction and, indeed, the mechanism of inactivation of free rhodanese by phenylglyoxal plus cyanide remain a mystery, despite the fact that the structural alterations in the protein have been elucidated in detail.

Gel filtration of a reaction mixture containing 6 mg of free rhodanese, cyanide, and [‘4C]phenylglyoxal showed that 1) the protein was not polymerized during inactivation and 2) there was no incorporation of 14C label into the protein product. As shown in Fig. 7, the inactive product is removed at the same position as free rhodanese. Further, all of the radioactivity and absorbance at 250 nm associated with the phenylglyoxal eluted together with the small molecular reaction products. The ultraviolet absorption spectrum of the inactivated protein was found to be the same as that of active enzyme, indicating once again that the reagent was not bound covalently to the enzyme.

Fractions I and III (Fig. 5) were analyzed extensively for peptide content and for correlations between individual arginyl residues and radioactivity. These studies included automated Edman degradation of the fractions and the analysis of chymotryptic peptides derived from them. The results demonstrated that the phenylglyoxal modification of peptides in Fractions I and III was scattered among several arginyl residues including those at positions 64, 146, 158, 175, 186, 205, and 229 in the polypeptide. A substantial amount of the 14C! label in Fraction I (-20%) was due to the reaction of Arg-186. In this case, however, the residue was located in a larger peptide extending from Ala-183 to Arg-229. Low levels of radioactivity were observed at Arg-64, Arg-146, and Arg-158. Although, as will be discussed later, the phenylglyoxal modification of arginines in rhodanese provides experimental difficulties that are characteristic of the reagent, all the results point to Arg-186 as the major site of reaction. In no case was the degree of labeling of any other arginyl residue greater than 10 to 15% of that observed at Arg-186.

Amino acid analysis of phenylglyoxal-inactivated free rhodanese showed no change in the value for any amino acid, within the limit of experimental error. Determination of sulfhydryl groups by reaction with Ellman reagent, however,

Inactivation of Free Rhodanese by Phenylglyoxal and Characterization of the Protein Derivative--In order to study chemical modifications of free rhodanese, it is necessary first to remove the covalently bound substrate sulfur from the enzyme (20). This is done by adding an excess of cyanide to the protein, thus forming the thiocyanate product and free rhodanese. Since free rhodanese is somewhat unstable, it is customary to add the modifying reagent immediately after cyanide treatment, and it was in this way that the alkylations with iodoacetate described above were preformed. After several trials, it was established that complete and rapid inactivation of free rhodanese was caused by amounts of phenylglyoxal nearly stoichiometric with the enzyme. The stoichi- ometry of these reactions at pH 7.8 and at 25°C was one of the first indications that some residue other than arginine was

5 10 15 20

TIME (min) FIG. 6. Inactivation of free rhodanese by reaction with phenyl-

glyoxal, benzoylformoin, or methylglyoxal at pH 7.8, 25”C, in the presence of cyanide. a, [E] = 1.25 x 10m4 M; [phenylglyoxal] = 1.33

being modified. The time courses of inactivation of free rho- x lo-’ M; 6, [E] = 1.67 X IO-’ M; [benzoylformoin] = 2.5 X lo-’ M; c,

[E] = 1.25 x 10 4 M; [methylglyoxal] = 1.33 X 10m4 M.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


50 100 150 200


FIG. 7. Gel filtration of reaction products from the inactivation of free rhodanese by [2-‘%]phenylglyoxal and cyanide on a column (2.5 x 45 cm) of Sephadex G-75 eluted with phosphate buffer, ionic strength 0.1, pH 7.8 at 4°C; flow rate, 50 ml/h. Fractions of 2 ml were collected; 100~~1 aliquots were assayed for radioactivity.

showed that approximately two of the four free sulfhydryl groups in rhodanese had been lost in the inactive product. This, and several other observations, suggested that the phenylglyoxal-promoted inactivation of free rhodanese might be due to oxidation of the active site Cys-247, possibly by formation of a disulfide bond with a nearby cysteine residue. The fact that about 90% of the enzymic activity could be restored by incubation with 1 M Na2S203, was consistent with earlier observations (20) that such treatment of oxidized rhodanese results in concomitant regain of enzyme activity and sulfhy- dry1 groups. Moreover, the products resulting from inactivation by any of the c*-keto aldehydes or benzoylformoin were identical. These indications led us to determine whether or not a disulfide bond could be formed in the enzyme under these conditions and, if so, which cysteines were involved.

In order to generate peptides in quantities sufficient for structural characterization, 70 mg of free rhodanese was inactivated as described above, and the protein was passed through a larger column (5.0 x 50 cm) of Sephadex G-75 to remove reaction products. The protein was then concentrated and the unreacted cysteines were carboxymethylated under denaturing conditions without prior reduction. In order to examine the possibility of disulfide exchange under basic conditions during column chromatography, a separate experiment was carried out in which, after inactivation, the reaction mixture was made 6 M in guanidine hydrochloride and carboxymethylated directly without separating the reaction product. These two experiments gave the same results, showing that no significant amount of disulfide exchange had occurred during gel filtration. The carboxymethylated rhodanese was then citraconylated, desalted, and lyophilized. Amino acid analysis of this rhodanese derivative gave 2 residues of carboxymethylcysteine/33,000 g of protein, a result consistent with the assumption that a disulfide bond had been formed.

The rhodanese derivative thus obtained was digested with trypsin and the Tc-peptides were separated by gel filtration on Sephadex G-50 as before (Fig. 8). Comparison of this elution profile with that in Fig. 1 revealed two principal differences: 1) the absorption at 280 nm of Fraction I (Fig. 8) was higher than that of I (Fig. l), and 2) the absorption at 280 nm of Fraction IV (Fig. 1) had almost disappeared in IV from

the present experiment (Fig. 8). In earlier tryptic digests of citraconylated rhodanese derivatives, Fraction I consisted of a pure peptide (Tc7, His-51 to Arg-110; or Tc7b, Asp-65 to Arg-llO), and Fraction II contained Peptide Tc17 with the active site cysteine 247 (Ala-230 to Arg-248). Fraction IV contained the Peptide Tc18 from Lys-249 to Arg-281, which includes 2 cysteines, Cys-254 and 263 (6, 7). In the present analysis of peptides from phenylglyoxal-inactivated rhodanese, amino acid analysis of I showed that it was not the expected pure peptide, Tc7 (or Tc7b). Automated Edman degradation of this fraction through several steps showed that it contained in addition to Tc7b two other peptides, Tc17 and Tc18. This result suggested that these latter two cysteine peptides were held together by a disulfide bond and so emerged from the column earlier than usual in the elution diagram. If this were the case, a simple reduction and carboxymethylation of the Fraction I mixture would be expected to restore those two cysteine peptides to their original positions in the elution profile without altering the behavior of Tc7b (Asp-65 to Arg-110). Moreover, alkylation with [‘“Cl- iodoacetate would pinpoint the cysteines involved.

Indeed, as shown in Fig. 9, three peaks were resolved by gel filtration on Sephadex G-50 of the fragments produced by reduction and [“‘Clcarboxymethylation of Fraction I (Fig. 8). Amino acid analysis of the three fractions identified them as Tc7b, Tc17, and Tc18 (Fig. 9); radioactivity was distributed equally between Tc17 and Tc18. These results proved that, in phenylglyoxal-inactivated free rhodanese, there is a disulfide bond between Tc17 and Tc18; that is, the active site Cys-247 forms a disulfide bond with either or both Cys-254 or Cys- 263.

In order to establish which of the 2 cysteines in Tcl8 was involved in disulfide bond formation with Cys-247, it was necessary to identify which of the 2 carboxymethylcysteines in Peptide Tc18 was radioactive. This was done both by isolation of chymotryptic peptides from Tc18 containing Cys- 254 and Cys-263 and by automated Edman degradation of the intact Tc18. The chymotryptic peptides of Tc18 were isolated as described in the first of this series of papers (Table XIX; (6)). Peptide Cl containing Cys-254 and Peptide C3 containing Cys-263 were found to be labeled to the extent of 70% and 30%, respectively. Furthermore, Edman degradation of Tc18 was performed through 15 cycles and the radioactivity

1 0.4 E c

8 - 0.3

t

iti z 0.2

2

8 gj 0.1 a


FIG. 8. Gel filtration of the fragments produced by tryptic cleavage of S-carboxymethylated, citraconylated, phenylglyoxal-inactivated free rhodanese (70 mg). Separation was on a column (2.5 x 185 cm) of Sephadex G-50 (superfine) eluted with 0.1 M NH,HCO:s; flow rate, 30 ml/h. Fractions of 6 ml were collected.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

The Active Site of Rhodanese

Tcl7 a 1’ Tcl8

- 700

-600 5

-500 6

i

-400 8 T E

-300 ,”

300 400 500 600 700


FIG. 9. Gel filtration of the products obtained from reduction and [‘4C]carboxymethylation of Fraction I (Fig. 8) on a column (2.5 x 185 cm) of Sephadex G-50 (superfine) eluted with 0.1 M NHdHCO:j; flow rate, 30 ml/h. Fractions of 6 ml were collected; 100.~1 aliquots were assayed for radioactivity.

of the residue released at each cycle was determined. The results, when corrected for the repetitive yield of the degradation, also showed that Cys-254 was approximately 70% labeled and Cys-263 was approximately 30% labeled. Thus, the phenylglyoxal:cyanide-promoted inactivation of free rhodanese is the consequence of formation of intramolecular disulfide bonds involving the sulfhydryl group of the essential Cys-247. In 70% of the molecules this bond involves Cys-254 and in the remaining 30% the bond is to Cys-263. The same results and the same ratio of disulfide bonds were observed in experiments with methylglyoxal, so that the nature of the alkyl or aryl substituent does not affect the course of the reaction. Control experiments proved that the disulfide bonds did not result from exposure to denaturing conditions (28) in 6 M guanidine hydrochloride.

It was of interest to determine whether an intramolecular disulfide bond is formed between Cys-254 and Cys-263 during the course of the reaction. Since these 2 cysteine residues are both in Tc18, the formation of a disulfide bond between them would not have altered the position at which these residues emerged from the Sephadex column. Sequence analysis of Fraction IV (Fig. 8) which should contain any Tc18 with a Cys-254-Cys-263 disulfide, showed less than 5% of peptide compared to that amount found in I (Fig. 8). This small amount might easily be accounted for by unreacted cysteine or by scission of disulfide bonds already formed between Peptides Tc17 and Tc18. Therefore, the disulfide bond between Cys-254 and Cys-263 is unlikely.

Another question that arises in this connection is whether the evidence leading to the inference of two different kinds of disulfide bond (70% between Cys-247 and Cys-254, and 30% between Cys-247 and Cys-263) could be the result of some kind of experimental artifact. It was conceivable, for example, that the radioactivity in Cys(Cm)-263 could have resulted from the loss of an unlabeled carboxymethyl group during reduction or other procedure, followed by reaction with [‘4C]iodoacetate. The abnormal behavior of Cys(Cm)-263 has been noted in the preceding paper (7). In order to examine such a possibility, [‘4C]iodoacetate was used for carboxymethylation of the unreacted cysteines immediately after the inactivation reaction. Edman degradation of the mixture of

peptides in Fraction I was carried out and the product of each cycle was counted for radioactivity. Cys-254 was found to be approximately 30% labeled and Cys-263 was approximately 70% labeled. These findings confirmed the earlier observations that both Cys-254 and Cys-263 are able to form disulfide bonds with Cys-247. To test the hypothesis that one of these two disulfide bonds might be formed much faster than the other, free rhodanese inactivated by phenylglyoxal-cyanide to the extent of 50% was analyzed. The same results were obtained; that is, the same two disulfide bonds were formed in the same ratio.

Considering all the evidence, we conclude that phenylglyoxal plus cyanide inactivate free rhodanese by oxidation of the active site Cys-247 to form intramolecular disulfide bonds with Cys-254 (70%) and Cys-263 (30%).

DISCUSSION

The results of [‘4C]iodoacetate inactivation experiments under mild conditions showed unequivocally that the site of alkylation of rhodanese is Cys-247. The demonstration that the active site cysteine is Cys-247 is consistent with the result of Blumenthal and Heinrikson (8) and also with the findings of Bossa et al. (29) for bovine kidney rhodanese. In the present work, however, near-stoichiometric levels of iodoacetate were employed, thus precluding secondary alkylations which have, in the past, led to some confusion regarding the identification of the active site cysteinyl peptide (30) and the number of functional cysteines per mol of enzyme (8, 20, 30).

The identification of SY of Cys-247 as the site of covalent attachment of the sulfane atom in sulfur-rhodanese is in accord with the x-ray crystallographic findings in which the persulfide linkage at Cys-247 was observed. Difference Four- ier analysis of cyanide-treated crystals indicated cleavage of the sulfane-SY-Cys-247 bond (9).

The pK’ value of the active site sulfhydryl group of Cys- 247 in free rhodanese is 7.8 as established by the pH profile of the data from inactivation studies with iodoacetate. Normal sulfhydryl groups have pK values from 7.2 to 10.2 (31), depending on the environment of these groups. The pK’ value for Cys-247 is in the lower range of normal sulfhydryl groups, thus suggesting that it is in a cationic environment (11, 31). This would be consistent with the early finding that cationic groups are in the active site of rhodanese (12). Previous kinetic studies (11) have established the pK’ value of the essential sulfhydryl group in rhodanese. thiosulfate complex to be 6.5. Since it is impossible to inactivate rhodanese by iodoacetate in the presence of thiosulfate, this pK’ value was simulated in the present work by studying the inactivation in the presence of 0.5 M benzenesulfonate, a substrate analog and inhibitor competitive with thiosulfate (20). Similar inactivation studies in the presence of 0.5 M butanesulfonate served as a control for general ionic effects, since this compound is a very poor inhibitor (20). In the presence of benzenesulfonate, the pK’ of this sulfhydryl group was shifted to 6.7, which was close to the value for that in the rhodanese. thiosulfate complex. In the presence of butanesulfonate, its pK’ was shifted to 7.05. These results suggest that about half of the pK shift in the rhodanese . thiosulfate and rhodanese. benzenesulfonate complexes is due to the ionic effect of these compounds. The remainder, perhaps is caused by the conformational change known to accompany substrate binding in the active site (32, 33).

From our study of the inactivation of sulfur-rhodanese by phenylglyoxal, we conclude that 1 arginyl residue, Arg-186, is essential for rhodanese activity. The complete protection against inactivation afforded the enzyme by high concentrations of substrate supports, but does not prove, the conclusion


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


that Arg-186 is at the active site. The use of phenylglyoxal as a general modifier of guanidino groups (27) is fraught with experimental difficulties. Substantial molar excesses of reagent are required for reasonable rates of reaction and, under these conditions, the arginyl side chains which are, by and large, exposed at the surface of the protein tend to react extensively and nonspecifically. Difference labeling methods such as employed in this work help to increase the selectivity of active site residue labeling, but do not allow for absolute discrimination between essential and nonessential groups. Moreover, under neutral or slightly alkaline conditions of pH, the phenylglyoxal reaction is reversed (27) so that one usually encounters a loss of adduct during the isolation of labeled peptides. This often confuses the quantitation of modification at any given arginyl residue as well as the identification of functionally essential guanidino groups. In the present study, the sites of modification were identified in Tc fragments (Fig. 5), the isolation of which required exposure of peptides to slightly alkaline conditions for 48 h. This strategy facilitated comparison with control digests (Fig. l), but the basic conditions of pH led to a loss of about 40% of the “‘C label in Arg- 186. Nevertheless, Arg-186 was, by far, the most radioactive of the arginines in the protein. Exhaustive analysis of other ‘C-labeled arginyl peptides failed to reveal any other arginine which could be interpreted as a major site of 14C! incorporation.

Kinetic studies of sulfur-rhodanese inactivated by phenylglyoxal to the extent of about 96% indicate that the loss of enzyme activity is due largely to inhibition of thiosulfate binding. This could be due to the loss of a positive charge required in the electrostatic interaction with substrate. How- ever, the inactivation by phenylglyoxal could also be explained by steric hindrance or by conformational changes since two bulky groups are introduced into the guanidino side chain.

The former interpretation seems more plausible based upon the x-ray crystallographic results which suggest the participation of Arg-186, Lys-249, and Thr-252 in substrate binding (9,lO). Although there is no chemical evidence for the involvement of a threonine residue in either binding or catalysis, amino groups have been implicated by inactivation studies with pyridoxal Y-phosphate (14). It could be that the modification of Lys-249 was responsible for the loss of activity, but no specific site of reaction was identified. A pK’ of 9.9 was calculated from kinetic studies of the pH dependence of the dissociation constant for the complex of thiosulfate with free rhodanese (11). This pK’ could be an ionization constant for water molecules associated with the cationic site or it could be that of Lys-249. It is also conceivable that the pK of Arg- 186 could be as low as 9.9 depending upon the special microenvironment of the active site.

The results described herein from chemical modification studies are compatible with the x-ray crystallographic findings (9, 10) in support of Arg-186 as one of the components of the cationic cluster involved in binding the anionic substrate. The distance between the guanidino group of Arg-186 and the sulfane atom in sulfur-rhodanese is 8 to 9 A. Two other arginine residues in the vicinity of the active site are Arg-248 and Arg-182, but these do not appear to react with phenylglyoxal. The fact that Arg-248 forms salt bridges with Glu-71 and Glu-193, and Arg-182 forms salt bridges with Glu-193 and Asp-180, might possibly explain their apparent inertness to reaction with phenylglyoxal. Arg-186 forms only one salt bridge, that being with Glu-193 (9).

In contrast to the inactivation of sulfur-rhodanese by phenylglyoxal via the usual modification of arginyl residues, the loss of activity of free rhodanese promoted by this reagent in the presence of cyanide has been correlated with the formation of disulfide bonds involving the active site Cys-247. Although

phenylglyoxal is fairly specific in its reaction with guanidino groups, this reagent and methylglyoxal are known to react reversibly with the sulfhydryl group of cysteine and many naturally occurring thiols (34, 35). However, the specificity of these reagents in promoting disulfide bond formation in rhodanese is remarkable and unique. Of all the carbonyl compounds tested, only phenylglyoxal, methylglyoxal, glyoxal, and benzoylformoin were effective as inactivators of the enzyme through oxidative disulfide bond formation. Phenyl- glyoxal has an advantage over the other reagents in that it reacts faster. The reason for this specificity is not entirely clear. Benzaldehyde and some other aromatic aldehydes have been shown (14) to inactivate rhodanese, but only with large excesses of reagents. The inactivations were accompanied by the loss of lysine residues. Efforts are currently underway to identify the product formed from phenylglyoxal during the oxidation of the enzyme and to define the role of cyanide in the reaction.

The inactive, oxidized, rhodanese product has been shown to consist of a mixture of molecules, 70% of which contain a disulfide bond between Cys-247 and Cys-254; in the remaining 30%, the bond is between Cys-247 and Cys-263. Others (36,37) have suggested that the active site cysteine is proximal to a nonessential sulfhydryl group. Our results establish the spatial proximity of the side chains of Cys-247 and Cys-254, and of Cys-247 and Cys-263 in free rhodanese. Furthermore, the distance between the side chains of Cys-247 and Cys-254 would appear to be shorter than that between Cys-247 and Cys-263. X-ray diffraction studies of sulfur-rhodanese (9, 10) show that the sulfur atom of Cys-247 is 7 A from that of Cys- 254 and 19 A from that of Cys-263. These distances would preclude the formation of the observed disulfide bonds without significant conformational changes in the enzyme. Since sulfur-rhodanese was used for the x-ray diffraction studies, one possible explanation is that a large conformational change accompanies removal of the sulfane sulfur atom bound to Cys- 247, bringing these 3 cysteine residues close enough to form intramolecular disulfide bonds in free rhodanese. The occur- rence of a gross conformational change during the catalytic cycle of rhodanese has been indicated by thermodynamic studies (32, 38) and established by circular dichroism, optical rotatory dispersion, and absorption studies (33). The other possible explanation is that the conformation of crystalline rhodanese differs considerably from that of the enzyme in solution.

The question regarding the existence of a substantial conformational difference between free rhodanese and sulfur- rhodanese has been addressed by x-ray crystallography and the results are at variance with those obtained by studies in solution. Little conformational change was observed by difference-Fourier analysis of crystals soaked in cyanide and washed with ammonium sulfate, even though cleavage of the persulfide linkage was clearly documented (9). In this experiment, however, it was not clear that the thiocyanate product had been removed from the active center since a site in that region was still occupied either by sulfate ion or by product. The crystal forces in sulfur-rhodanese may, therefore, be strong enough to prevent diffusion away of the thiocyanate so that restoration of the conformation of free rhodanese is prevented. This might explain why a crystalline form of the free enzyme has not been obtained. It can also be argued that the novel oxidation of rhodanese by phenylglyoxal is the result of an altered conformation induced by the reagent. This seems less likely since methylglyoxal is equally effective in promoting the reaction.

The cause(s) of the apparent discrepancies between the x- ray structural model of the active site of sulfur-rhodanese and


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


the inferences drawn from measurements of this derivative and free rhodanese in solution therefore remains an interesting question. Attempts to crystallize the mixture of inactive disulfide-bonded rhodanese derivatives are in progress in the hope that a sample suitable for crystallographic analysis might be prepared. Such studies should provide a view of the active site which differs considerably from that seen in sulfur-rhodanese, a view which may correspond more closely to the situation in the free enzyme.

An expanded formal mechanism for rhodanese action was proposed in 1974 by Schlesinger and Westley (11) based on all the information obtained up to that time. Ploegman (9) has subsequently described a very similar mechanism modified slightly in view of the three-dimensional model derived from

x-ray diffraction studies. The chemical mechanism of rhodanese catalysis proposed in Scheme 2 was formulated on the basis of the most recent results from crystallographic analysis and from studies of the enzyme in solution. This mechanism can be described in the following way. Rhodanese contains a cationic binding site, represented as EHs’+, which may consist of Arg-186 and Lys-249 together. Solvent H20 molecule(s) might be coordinated with this cationic site. The enzyme. thiosulfate complex forms when S203’- displaces one Hz0 ligand. This step depends upon a pK’ of 9.9, with the protonated enzyme binding the substrate much more strongly than the deprotonated form (11). In the enzyme. thiosulfate complex, the S-S bond of S203’- is polarized by the electrophilic site (presumably identical with the cationic site), leaving the

IUIO-EH~+~-OH~ H+

I 5

4 S-H

Ii $039

R~o-EH~+~-SO~-~ H+

I / s e

S-H

R~o-EH~+~-OH~

I

H+ \

\ S-SH

k CN-

R~o-EH~+~-OH~ I 1 f

I n- S-SH CN

SCN-

R~o-EH,+~-OH,

I

H+

- SH

R~o-EH~+~-OH~ H+ Rho-EH2+QHe

I .

P I Se Se

11 s2o3-2

Rho-EH,+2-sop

I P p-2 s

I R~o-EH~+~-SO~-~

I s-8

R~o-EH~+~-OH, H+ R~o-EH~+~-OH~

I \

\ I S-s-

CN-

I

9

I? SCN-

R~o-EH~+~-OH,

I Se

SCHEME 2


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


sulfane sulfur neutral or even slightly positively charged. An enzymic nucleophile, the thiolate anion of Cys-247, then attacks this sulfur and displaces sulfite to form the sulfur- rhodanese, a persulfide type of intermediate stabilized in the nonaqueous microenvironment of the active site. This is the rate-limiting step at maximum velocity and is dependent upon a pK’ of 6.5, with the protonated form being unreactive (11). That this pK’ relates to Cys-247 in the enzyme. thiosulfate complex has been shown in the present work. The sulfite product left coordinated to the cationic site upon substrate cleavage is then displaced by a water molecule. The x-ray crystallographic evidence shows that the persulfide in sulfur-rhodanese is stabilized by hydrogen bonds between the sulfane sulfur as acceptor and a number of NH groups of the main chain and the hydroxyl group of Thr-252 as donors (9). The reaction between sulfur-rhodanese and cyanide is very fast since no kinetically significant complex has been detected (12, 39), and the second order rate constant approaches the diffusion-controlled limit (11). The transition state for this reaction must, therefore, resemble an outer sphere complex of cyanide anion with the enzymic cationic site (11). The products of the reaction with cyanide are the free enzyme and thiocyanate. This reaction depends on pK’ values of 5.9 and 9.4, the least protonated form being unreactive (11). The pK of 9.4 may be represented as an ionization of a water ligand similar to that in the free enzyme. The pK’ of 5.9 is much higher than would be expected for a normal persulfide, but for an enzymic persulfide the normal value might well be shifted by its unusual chemical environment. This mechanism does not provide an explicit role for the hydrophobic region, but it is suggested that the hydrophobic microenvironment of the active site must be important both in increasing the strength of the electrostatic interaction of substrate binding and in stabilizing the hydrogen bonding structure of the persulfide intermediate.

REFERENCES 1. Westley, J. (1973) Adu. Enzymol. 39, 327-368 2. Sorbo, B. H. (1975) in Metabolic Pathways (Greenberg, D. M.,

ed) Vol. 7, pp. 433-455, Academic Press, New York 3. Westley, J. (1977) in Bioorganic Chemistry (Van Temalen, E. E.,

ed) Vol. 1, pp. 371-390, Academic Press, New York 4. Horowitz. P., and DeToma. F. (1970) J. Biol. Chem. 245.984-985 5. Blumenthal,‘K. M., and Heinrikson; R. L. (1971) J. B&l. Chem.

246, 2430-2437 38. Leininger, K. R., and Westley, J. (i968) J. Biol. Chem. 243,

1892-1899 6. Weng, L., Russell, J., and Heinrikson, R. L. (1978) J. Biol. Chem. 39. Westley, J., and Heyse, D. (1971) J. Biol. Chem. 246, 1468-1474

7.

8.

9.

10.

11. 12. 13. 14.

15.

16.

17. 18. 19. 20. 21.

22. 23.

24. 25.

26.

27. 28.

29.

30.

31. Webb, J. L. (1966) Enzyme and Metabolic Inhibitors, Vol. 2, pp.

32. 33.

635-653, Academic Press, New York Wang, S.-F., and Volini, M. (1973) J. Biol. Chem. 248,7376-7385 Volini, M., and Wang, S.-F. (1973) J. Biol. Chem. 248, 7386-7391 Schubert, M. P. (1935) J. Biol. Chem. 111, 671-678 Takahashi, K. (1977) J. Biochem. (Tokyo) 81,395-402 Guido, K., and Horowitz, P. M. (1976) Bioph.ys. J. 16, 203a Pensa, B., Costa, M., Pecci, L., Cannella, C., and Cavallini, D.

(1977) Biochim. Bioohrs. Acta 484. 368-374

34. 35. 36. 37.

253,8093-8101 Russell, J., Weng, L., Keim, P. S., and Heinrikson, R. L. (1978) J.

Biol. Chem. 253,8102-8108 Blumenthal, K. M., and Heinrikson, R. L. (1972) Biochim. Bio-

phys. Acta 278,530-545 Ploegman, J. H. (1977) Ph.D. dissertation, University of Gronin-

gen Ploegman, J. H., Drenth, G., Kalk, K. H., Hol, W. G. J., Heinrik-

son, R. L., Keim, P., Weng, L., and Russell, J. (1978) Nature 273, 124-129

Schlesinger, P., and Westley, J. (1974) J. Biol. Chem. 249,780-788 Mintel, R., and Westley, J. (1966) J. Biol. Chem. 241, 3386-3389 Horowitz, P., and Westley, J. (1970) J. Biol. Chem. 245, 986-990 Cannella, C., Pecci, L., Costa, M., Pensa, B., and Cavallini, D.

(1975) Eur. J. Biochem. 56,283-287 Karrer, V. P., and Segesser, A. V. (1935) Helu. Chim. Acta 18,

273-275 Ruggli, P., Dahn, H., and Fries, P. (1946) Helu. Chim. Acta 29,

302-313 Sorbo, B. H. (1953) Acta Chem. Stand. 7, 1129-1136 Westley, J., and Nakamoto, T. (1962) J. Biol. Chem. 237,547-549 Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 Wang, S.-F., and Volini, M. (1968) J. Biol. Chem. 243,5465-5470 Long, C., ed (1961) Biochemists’Hundbook, p. 32, Van Norstrand

Co., Inc., New York Dixon, M. (1953) Biochem. J. 55, 161-171 Schmitt, D. E., Jr., and Westheimer, F. H. (1971) Biochemistry

10, 1249-1253 Mintel, R., and Westley, J. (1966) J. Biol. Chem. 241, 3381-3385 Spackman, D. H., Stein, W. H., and Moore, S. (1960) J. Biol.

Chem. 235,648-659 Heinrikson, R. L., Sterner, R., Noyes, C., Cooperman, B. S., and

Bruckmann, R. H. (1973) J. Biol. Chem. 248,2521-2528 Takahashi, K. (1968) J. Biol. Chem. 243,6171-6179 Baillie, R. D., and Horowitz, P. M. (1976) Biochim. Biophys. Acta

429, 383-390 Bossa, F., Cannella, C., Federici, G., Barra, D., and Pecci, L. (1973)

FEBS Lett. 29, 170-172 DeToma, F., and Westley, J. (1970) Biochim. Biophys. Acta 207,

144-149


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

L Weng, R L Heinrikson and J Westleydisulfide bonds in the active site promoted by phenylglyoxal.

Active site cysteinyl and arginyl residues of rhodanese. A novel formation of

1978, 253:8109-8119.J. Biol. Chem.

http://www.jbc.org/content/253/22/8109Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/253/22/8109.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/253/22/8109

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;253/22/8109&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/253/22/8109

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=253/22/8109&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/253/22/8109

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/253/22/8109.full.html#ref-list-1

http://www.jbc.org/

the journai. of biological chemistry november 25, pp. … · the journai. of biological chemistry...

Documents