ARCHIVES

OF BIOCHEMISTRY

On the Physical

AND

Properties

Sulfohydrolase E. J. SAMPSON,

Department

169, 372-383 (1975)

BIOPHYSICS

and Mechanism I I from Aspergihs

E. V. VERGARA, J. M. FEDOR, BENKOVIC *

of Chemistry,

of Action

oryzae l M. 0. FUNK,

The Pennsylvania State University, 152 Dave.v Laboratop, Pennsylvania 16802 Received November

of Arylsulfate

AND

S. J.

University

Park,

11, 1974

Sedimentation equilibrium studies on arylsulfate sulfohydrolase II (EC 3.1.6.1) from Aspergillus oryzae under nondissociating conditions have resulted in a revised molecular weight of 94,900 * 7100. Sedimentation equilibrium and gel electrophoresis data collected in the presence of the dissociating agents, urea and sodium dodecylsulfate demonstrate that the native enzyme is composed of two identical subunits as suggested by previous studies employing an irreversible inhibitor. The pH dependencies of the kinetic parameters V and V/K,,, for the enzymic hydrolysis of 4-nitrophenyl sulfate indicate that two groups of pK, 4.7 and 6.0 control the activity of the enzyme. The product inorganic sulfate was shown to be a linear competitive inhibitor of the enzyme at pH 4.0, implying that it is a last released product along the reaction pathway. Inhibition by the phenol product was not observed. Enzymic hydrolysis of 4-nitrophenyl sulfate in I80 enriched water revealed that one atom of solvent oxygen is incorporated per molecule of inorganic sulfate, which is consistent with a mechanism featuring sulfur-oxygen bond cleavage. Evidence is presented based on stopped-flow kinetics, partitioning experiments in the presence of amine nucleophiles, and “0 exchange studies that collectively suggest that the breakdown of a covalent sulfuryl enzyme intermediate probably is not the rate-limiting step along the reaction pathway. The substrate specificity of the enzyme was examined by testing a variety of sulfate and phosphate esters as inhibitors of the hydrolysis of 4-nitrophenyl sulfate. The Cbz-L-Phe-LTyrosine-O-sulfate methyl ester serves as a substrate for the enzyme. Apparently substrate activity requires an aromatic sulfate ester whose binding is enhanced by incorporating the aromatic moiety in a hydrophobic matrix.

bonded subunits (2). Benkovic et al. (31, employing the procedure of Cherayil (4), obtained a preparation of the isoenzyme which appeared homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. Recently, the purified enzyme was reacted with the irreversible inhibitor onitrophenyl oxalate, the first reported active site titrant for arylsulfatases (5). The results of this titration experiment suggested the presence of two active sites per molecule of enzyme. The previous experiments also included the determination of a structure-reactivity correlation for the enzyme catalyzed hydrolysis of a series of substituted phenyl sulfate esters. It was found that V was

Previous work on the arylsulfate sulfohydrolases (EC 3.1.6.1) from Aspergillus oryzae focused primarily on isolation procedures from commercially available preparations known as “Takadiastase” and Cherayil and Van Kley (l), “Clarase.” growing the microorganism on moist wheat bran, observed three distinct isoenzymes which varied in proportion to the cultural conditions. In a preliminary investigation on a partially purified preparation of these isoenzymes, Drnec concluded that isoenzyme II consisted of a single polypeptide chain and did not contain noncovalently ‘This investigation was supported by NIH grant GM 13306. 2 To whom correspondence should be sent. 372 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.

PROPERTIES

OF ARYLSULFATE

insensitive to the pK, of the departing phenol (P x 0) suggesting among others a reaction scheme involving rate-determining breakdown of a sulfuryl enzyme intermediate. The present investigation of this enzyme was initiated to (1) seek additional physical evidence for the subunit structure of the purified protein; (2) gain data via kinetic and tracer techniques that would bear on the viability of the sulfuryl enzyme concept; and (3) further define the substrate specificity of the enzyme. EXPERIMENTAL

PROCEDURES

All melting points are uncorrected. D,O (99.8’%, Diaprep), HZ’“0 (Biorad), and twice-distilled deionized water were employed as solvents. Reagent grade salts, solvents, and acids (Fisher, Baker) were used without further purification, except where noted. Descending paper chromatograms were run on Whatman No. 1 paper in 1-butanol/acetic acid/water (50:12:25) and spots detected with 0.25a ninhydrin and uv visualization whenever applicable. In situ hydrolysis of the chromatographed product was carried out in dioxane/HCl (1:6) at room temperature and the hydrolysis products detected with Burma’s spray reagent for inorganic sulfate (6) and Barton’s reagent for the free phenolic hydroxyl (7). Methoxyamine hydrochloride was recrystallized and aniline distilled prior to use. L-Tyrosine (Biochemical Nutritional Laboratory) and N-carbobenzoxy-L-phenylalanine (Sigma) were homogeneous by paper chromatography and used without further purification. Nuclear magnetic resonance spectra in D,O were measured on a Varian Associates A-60 spectrometer using sodium 2,2-dimethyl-2.silapentane-5.sulfonate as the internal standard. Ultraviolet spectra were obtained on a Cary 14 recording spectrophotometer. Mass spectra were measured on an MS-902 AEI spectrometer. Materials. The potassium salts of n-glucosel-phosphate, o-glucose-6-sulfate, indoxyl sulfate, phenolphthalein disulfate, estrone-3-sulfate, and 4. nitrophenyl sulfate (P-NPS)~ were obtained from Sigma and used without further purification. The sodium salts of hexyl-, decyl-, dodecyl-l-sulfates and androstenolone-3-sulfate were kindly donated by Dr. R. 0. Mumma of the Pesticide Research Laboratory, The Pennsylvania State University. The brucine salt of D-galactose-6-sulfate was obtained from Miles Laboratory. L-Tyrosine-O-sulfate was prepared by the procedure of Dodgson et al. (8). The product was recrystal3 Abbreviations fate; and DTNB,

used: p-NPS, 4-nitrophenyl sul5,5-dithiobis(2-nitrobenzoic acid).

A. ORYZAE

373

lized twice from anhydrous methanol and ether: R, 0.26; uv A,,, 265 (C = 398, 1 N NaOH); (D,O) 63.28 (d. 2H, -CH,-), 4.39 (t, lH, -CH-), 7.10 (2d, 4H. phenolic); mp > 290°C decomposed. L-Tyrosine methyl ester hydrochloride was prepared by literature methods (9). R, 0.60; uv X,,. 293 (c = 2250, 1 N NaOH), 240 (e = 9950, 1 N NaOH); (D,O) d 3.20 (d, 2H, -CH,-), 3.85 (s, 3H, -CH,-), 4.35 (t, lH, -CH-), 7.02 (2d, 4H, phenolic): mp 194-195°C. N-Carbobenzoxy-L-phenylalanine-L-tyrosinemethyl ester was prepared by the general method of Woodward et al. (10). N-Carbobenzoxy-L-phenylalanine, 500 mg (1.67 mmol) was dissolved in 28 ml of acetonitrile and then cooled to 0°C. Triethylamine, 0.23 ml (1.67 mmol) and Woodward’s Reagent K’, 423 mg (1.67 mmol) were added with stirring for 1 h at 0°C. Then 387 mg of L-tyrosine methyl ester hydrochloride (1.67 mmol) was added and the mixture stirred overnight at room temperature (15 h). The acetonitrile was evaporated in U~CUOand the residue was dissolved in ethyl acetate/water (2O:lO). The ethyl acetate layei was washed twice with distilled water and evaporated to dryness. The residue was washed with 0.5% sodium bicarbonate, with water, and redissolved in ethyl acetate. Removal of the ethyl acetate under vacuum gave a white residue: R, 0.91; uv hIlax 293 (C = 2525, 1 N NaOH), 240 (c = 32,200, 1 N NaOH); (acetone-d,) 6 2.90 (d, 2H, -CH,-), 3.04 (d, 2H, -CH,-), 3.62 (s, 3H, -CH,-), 4.50 (t, lH, -CH-CH,-phenyl), 4.80 (s, 2H. -CH,-O-C-). 6.85 (2d, 4H, phenolic). 7.20 (s. 5H, phenyl), 7.28 (s, 5H, phenyl); mp 136-137°C. Hydrolysis of 10 mg of the peptide in 6 N hydrochloric acid by refluxing for 20 h, followed by chromatography of the hydrolysate, gave spots corresponding to L-tyrosine (R, 0.48) and L-phenylalanine (R, 0.61) (11). N-Carbobenzoxy-L-phenylalanine-L-tyrosineO-sulfate methyl ester was synthesized by a modification of the method described by Reitz et al. (12). Fresh pyridine sulfur trioxide (Aldrich, 645 mg) and N-carbobenzoxy-L-phenylalanine-L-tyrosine methyl ester (200 mg) were suspended in freshly distilled pyridine in a 25ml, round-bottomed flask fitted with a drying tube. The reaction mixture was stirred under reflux at 115°C for 5 h. The hot solution was then transferred to a 50-ml, round-bottomed flask and the pyridine removed in uacuo. The residue was dissolved in cold anhydrous methanol and neutralized with cold 1 N methanolic sodium hydroxide solution. The precipitated sodium sulfate was removed by filtration, and the filtrate was concentrated to dryness in vacua. The residue was redissolved in a minimum amount of cold anhydrous methanol and precipitated with anhydrous ether. The product was reprecipitated twice in cold anhydrous methanol-ether and dried under vacuum at room temperature: R, 0.64; uv X,,. 260 (D,O) d 2.80 (d, 2H, (C = 564, 1 N NaOH);

374 -Ok), -CH,-),

SAMPSON

3.05 (d, 2H, -CH,-), 3.75 (s, 3H, 6.90 (2d, 4H, phenolic), 7.20 (s, 5H, phenyl), 7.30 (s, 5H, phenyl); mp >250°C decomposed. In situ hydrolysis in dioxane-hydrochloric acid of a chromatographed sample gave positive results for inorganic sulfate (R, 0.64) and phenolic OH (R, 0.63). Arylsulfate sulfohydrolase II was purified according to the procedure reported previously (3). Protein concentrations were determined by the method of Lowry et al., with crystalline bovine serum albumin as the standard (13). Relative activity measurements for enzyme stock solutions were determined by the method of Huggins and Smith (14) as modified by Benkovic et al. (3). Specific activity is defined as pmol of 4nitrophenol produced/min/mg of protein at 37.5% under the assay conditions. The homogenous protein exhibited a sp act of 3.1 (pH 5.8). A partially purified preparation, sp act 0.9, was employed only in the stopped-flow and ‘*O exchange experiments. Stock solutions of both preparations were stored in acetate buffer at pH 4.8, I = 0.05, -5”C, at protein concentrations between 0.1 and 1.0 mg/ml. Physical methods. Polyacrylamide gel electrophoresis was carried out at pH 8.6 using a discontinuous Tris-glycine buffer system as described by Ornstein and Davis (15). Gels were fixed and stained for protein with Coomassie brilliant blue (16). Conventional sedimentation equilibrium studies were performed with the Spinco Model E ultracentrifuge equipped with an ultraviolet scanner operated at 280 nm (17). All runs were carried out at 5°C using approximately 2 mm solution columns. Actual calculations on the raw data obtained from the scanner traces were executed in the IBM 360/67 computer with a Fortran IV program. By using a least squares regression analysis, the slope of the plot of In C against X’ and the standard error of the estimate from the slope were computed. The apparent molecular weight of the enzyme was calculated from the slope. Partial specific volumes of 0.722 and 0.726 from the amino acid analysis were used in all the calculations (3). The latter value was determined by assuming that aspartic acid and glutamic acid were present as asparagine and glutamine. All samples were dialyzed against the appropriate solvents for at least 24 h prior to ultracentrifugal analysis. Kinetics. The hydrolysis of the aromatic sulfate esters employed in this study were monitored spectrophotometrically with a Gilford 240 spectrophotometer at the appropriate wavelength (3). All kinetic experiments were maintained at 37.5”C (hO.1”) with a Haake circulating water bath. In atypical determination 0.050 ml of enzyme stock solution was added to 0.35 ml of buffer in a cuvette and placed in the thermostated cell compartment of the spectrophotometer. After 7 min. 0.10 ml of preequilibrated substrate solution was added and absorbance readings taken at a convenient time interval. Experiments

ET AL. with added inhibitor or nucleophile (aniline or methoxyamine) followed an identical procedure with the desired reagent added to the buffer solution. The buffers utilized were acetate (0.2 M, pH 3.5-5.5) and Tris-maleate (0.2 M, pH 6.0-8.0). All kinetic runs were carried out at I = 0.2, NaCl. The pH of the reaction solution at the completion of the kinetic experiment was determined to +0.02 pH units on a Radiometer Model 22 pH meter with a model pH 630 Pa scale expander. Initial velocities were determined from the slopes of straight lines obtained from plots of absorbance readings against time. Values for the kinetic parameters V and V/K, for the substrate p-NPS were statistically evaluated using the program HYPER described by Cleland (18). A minimum of eight substrate concentrations was employed for each analysis. Values employed for initial velocities in constructing the pH-rate profile were obtained from extrapolations to zero buffer. In the absence of a convenient method for directly measuring the hydrolysis of the compounds listed in Table II, each was screened as a potential inhibitor of the enzyme catalyzed hydrolysis of p-NPS. The inhibition constant, K,, was determined directly from Dixon plots of l/u against i for two substrate concentrations. Maximum differences in the slopes of the lines were achieved by working at substrate concentrations of 0.25-1.5 times the K, value and at inhibitor concentrations up to 1.3 times the K, value. In the cases of the competitive inhibitors, estrone-3sulfate and N-carbobenzoxy-L-Phe-L-Tyr-O-sulfate, their hydrolysis in the absence of p-NPS was monitored spectrophotometrically at the appropriate wavelength by quenching aliquots with 1.0 N sodium hydroxide at selected time intervals. Pre-steady state kinetics were investigated with a Durrum Gibson stopped-flow apparatus. The syringes, mixing chamber, and optical cell were thermostated at 25.O”C (+O.l”). Experiments were conducted at pH 4.02, 5.40, and 7.60 (I = 0.2). The substrate employed in this study was 2-chloro-4nitrophenyl sulfate at concentrations ranging between 5 x lo-’ and lo-’ M. At pH 4.02, production of 2chloro-4.nitrophenol was monitored at 318 nm (e = 8,360). At pH 5.40 and 7.60, production of the corresponding phenolate ion was monitored at 402 nm (e = 10,506 and 17,060, respectively). Prior to each set of determinations the pH and ionic strength of the enzyme solution were adjusted by dialysis to that of the substrate solutions. The active site concentration of the enzyme was calculated as the moles of subunits (molecular weight, 47,000) multiplied by the ratio of sp act (0.91/3.1). In the above initial velocity and stopped flow kinetic runs, at least 85% of the enzyme activity remained after dialysis, in accord with our earlier observations on the pH-stability of the enzyme over the pH range 4.0-7.6. I80 Tracer experiments. Four experiments were

PROPERTIES

OF ARYLSULFATE

conducted to test for ‘“0 incorporation from solvent into inorganic sulfate or substrate in the presence and absence of enzyme. Sample I was prepared by adding 200 Kmol of disodium sulfate and 0.3 ml of enzyme stock solution to 2 ml of ‘“0 enriched acetate buffer, pH 4, I = 0.2. This solution had an I80 enrichment of 4.3 atom % and was allowed to incubate for 72 h at 27°C. The turnover rate of the enzyme was approximately 0.05 mol/min under these conditions and over 85% of the initial activity was retained over this time interval. In control experiments: Sample II was prepared from 150 pmol of p-NPS incubated with the enzyme under the conditions of Sample I; Sample III from 200 pmol of inorganic sulfate incubated without enzyme under the conditions of Sample I; and Sample IV from p-NPS hydrolyzed to completion in 2 ml of 5.0 atom % I80 enriched acetate buffer, pH 4, 24 h, 100°C. Each solution was then concentrated under vacuum to 0.5 ml and the pH adjusted to pH 1 with 1 N hydrochloric acid. The precipitated protein was removed by centrifugation. Calcium chloride (220 pmol) was added to the decantate and the resulting precipitate (CaSO,) collected by centrifugation. The calcium sulfate was converted to the ammonium salt by dissolving the precipitate in the minimum amount of 0.5 M ammonium chloride and washing the solution through an Amberlite IR-120 column (1 x 5 cm) in the ammonium form. Each solution was evaporated to a volume of 0.5 ml and anhydrous ethanol was added dropwise until precipitation was complete. The ammonium sulfate was collected by centrifugation, washed several times with cold absolute ethanol, and dried at 100°C. The oxygens of ammonium sulfate were converted to carbon dioxide essentially by the method employed by Boyer et al. for inorganic phosphate (19). A small sample of ammonium sulfate (2 mg) was heated with guanidine hydrochloride (10 mg) in a vacuum line and the carbon dioxide that evolved was collected. The relative isotopic abundances occurring in the carbon dioxide were determined in an MS-902 AEI mass spectrometer by measuring peak heights directly from the instrument’s collector. Carbon dioxide isolated from Sample (III) was run as a standard prior to the other determinations. Trapping experiments. An experiment to trap sulfite as a product of the enzymic hydrolysis of aromatic sulfate esters was conducted as follows: 0.05 ml of enzyme stock solution was added to 0.35 ml of a solution containing 1.0 pmol of 5,5-dithiobis(2nitrobenzoic acid) (DTNB) and 0.5 Fmol of p-NPS at pH 4.8 (20). After incubation for 20 min at 37.5”C, the change in absorbance at 412 nm corresponding to the production of sulfite, was measured. The approximate rate of turnover for the enzyme under these conditions was 0.02 pmollmin. A control experiment revealed that DTNB had no detectable effect on the catalyzed rate of hydrolysis of the substrate.

A. ORYZAE

375

An experiment to trap intermediate quinone species was performed as follows: 0.10 ml of enzyme stock solution was added to 0.9 ml of a solution containing 4 pmol of sodium borohydride (21) and 0.2 rmol of 2-chloro-4-nitrophenyl sulfate. Two control experiments were also run; the first without sodium borohydride and the second without substrate. Each sample was incubated for 30 min at pH 7.6, 0°C. The three samples then were exhaustively dialyzed against acetate buffer, pH 4.8, I = 0.05, and the enzyme activity of each sample measured as described above. RESULTS

Molecular weight. A summary of the results of studies on the gel electrophoretic behavior of purified arylsulfate sulfohydrolase II follows. The protein exhaustively dialyzed against acetate buffer at pH 4.04 appears to be homogeneous on the gel electrophoregram whereas samples pretreated with 0.4% dodecyl sulfate or 8 M urea in the same buffer show two distinct bands. Protein dialyzed against Tris-acetate buffer at pH 7.61 and pretreated with 0.4% dodecyl sulfate or 8 M urea reveal only one band. Equilibrium sedimentation experiments on arylsulfate sulfohydrolase II in 0.05 M acetate buffer, pH 4.83, yielded a linear plot of In C against X2 to 50.8 cm2 from which an apparent molecular weight was computed from duplicate determinations. Figure 1 presents data obtained from equilibrium runs performed in 0.05 M acetate buffer, pH 4.04, and again in the same buffer prepared in 8 M urea. From the linear relationship observed in the presence of urea, an apparent subunit molecular weight was calculated. The upward curvature in the plot for buffer alone is indicative of a nonuniform molecular weight distribution throughout the cell suggesting a system involving an association-dissociation equilibrium (21). Although a weight average molecular weight could be computed from this data, its significance is questionable. In 0.05 M Tris-acetate buffer at pH 7.61, a linear relationship between In C and X” to 51.2 cm2 was noted. However, in the presence of 8 M urea a parabolic graph was found indicative of a partial dissociation of the enzyme into subunits. Values of the molecular weight measurements on the enzyme under the pretreatment conditions described above are given

376

SAMPSON

ET AL.

240 LrlC 200 I60

0.40 462

46.6

470

47.4

47.6

46.2 466 X2(d)

49.0

494

496

50.2

50.6

FIG. 1. Sedimentation equilibrium data for arylsulfate sulfohydrolase II (0.450 mg per ml) in 0.05 M acetate buffer, pH 4.04 (A) and in 0.05 M acetate buffer + 8 M urea, pH 4.04 (O), at 15,000 rpm for 20 h and 5°C.

in Table I. Molecular weights of 40,000 and 86,000 with an error of *lo’% were estimated from the above dodecyl sulfate gel electrophoresis studies employing the appropriate marker proteins (5). Kinetics. The effect of pH on the kinetic parameters V and V/K, for the enzymic hydrolysis of p-NPS is shown in Fig. 2. The kinetic scheme that describes the observed pH dependence is E”’

E”+S 11

K,,,)I Ii E”-I

kc,, =

k_,(lim)

(2)

(3) that V and on

‘S

-h!!i”‘~,

-

k,,,(lim)

1 + adKalEs + K,,EslaH

E” + p, + p,

(1)

11

Kc,,,, ( !! E”-‘S

where E is enzyme, S is substrate, ES is the adsorptive enzyme-substrate complex, P 1 and P, are products of the reaction of substrate with enzyme, n represents the net charge on the active form of the enzyme, and (lim) refers to the limiting values which would be obtained were all of the enzyme in the active form. The K, values are acid dissociation constants for the ionizing groups effecting catalytic activity. Alberty and Massey (22) have treated this scheme in detail and shown that

an acid of pK,,,, = 6.3. Similarly it was found that VIK, depends on an acid of pKasE = 6.1. Detection of a base corresponding to PK,,~:~ was not feasible due to enzyme inactivation below pH 4. Values of V(lim)/K,(lim) = 5.3 x lo4 min-’ and V(lim) = 12 x lo3 ~mol.min’ were employed in the solution of equations (2) and (3).

The pre-steady state kinetics at three points along the pH-V profile were examined using the stopped-flow technique and employing 2-chloro-4-nitrophenyl sulfate

PROPERTIES TABLE MOLECULAR

OF ARYLSULFATE

I

WEIGHT MEASUREMENTS ON ~YLSULFATE SULFOHYDROLASE II

Molecular

Solvent

weight”

53,206 zt 2400

0.05 M acetate buffer + 8 M urea, pH 4.04 0.05 M acetate buffer, pH 4.83b 0.05 M Tris-acetate buffer, pH 7.61

94,900 * 7100 91,300 * 4700

“The average error in the molecular weight was evaluated from the average of molecular weights calculated from values of 0.122 and 0.726 for the partial specific volume. b The average error is based on duplicate determination.

377

A. ORYZAE

reproduced from a typical run is shown by the solid line in Fig. 3. The plot of percent transmittance against time appears to be linear for 2 s, beginning at 100% Tat time zero. Replots of absorbance against time for similar experiments were linear for 14 s, intersecting zero absorbance at time zero. Under the conditions of the experiment, a “burst” of approximately 9 to 25% T (dependent upon changes in the molar absorptivity with pH) should be observed if rapid accumulation of a covalent sulfuryl enzyme intermediate and concomitant expulsion of 2-chloro-4-nitrophenol occurred. For example at pH 5.4, the molar absorptivity of the 2-chloro-4-nitrophenolate ion at 402 nm is 10,500 and the active site concentration of the enzyme is approximately 3.5 x 1Om6M (see Experimental). of 15% T should be Ideally a “burst” observable prior to the normal catalyzed hydrolysis of the substrate provided the mole fraction of active sites titrated approaches unity. An alternative kinetic approach to detect a transient sulfuryl enzyme species was based on partitioning of such a species between added nucleophiles and water. Measurement of the initial velocities for enzyme catalyzed hydrolysis of 2-chloro-4nitrophenyl sulfate in the absence and presence of 0.02 M aniline or methoxyamine at pH 4.8 gave identical rates.

PH

FIG. 2. The V-pH (01 and V/K,-pH (A) profiles for the hydrolysis of p-NPS by arylsulfate sulfohydrolase II at 37.5%. The solid lines are the theoretical curves calculated from equations utilizing (2) and (31 employing values in the text. The error bounds represent the standard errors as determined from program HYPER.

as substrate. A rapid initial decrease in the percent transmittance, i.e., a “burst,” prior to the normal turnover catalysis of the substrate was not observed at pH values 4.0, 5.4 and 7.6 and at substrate concentrations varying between 5 x lo-” and 10m4 M. The enzyme was not irreversibly inactivated during the runs since ca. 85% of the sp act of the enzyme remained after recovery and dialysis. An example of the stopped-flow data

Time

(secl

FG. 3. Data obtained from the stopped-flow experiment for the catalyzed hydrolysis of 2-chloro-4nitrophenyl sulfate by arylsulfate sulfohydrolase II at 25.O”C, pH 5.4 acetate buffer, [EO] = 3.5 x 1Om6M and [S,] = 10e3 M. The solid line is the actual plot of % transmittance against time observed on the stoppedflow instrument and the broken line represents the ideal “burst” height as calculated in the Results section

378

SAMPSON

Inhibition. Of the two products, inorganic sulfate and the corresponding phenol, released by the enzyme catalyzed hydrolysis of substituted aromatic sulfate esters, only inorganic sulfate revealed detectable inhibition. Utilizing 2-chloro-4nitrophenyl sulfate as the variable substrate, inorganic sulfate showed a linear competitive type of inhibition at pH 3.97 with Ki = 12.5 * 0.05 x 10m3 M-‘, Fig. 4. Inhibition by inorganic sulfate was also observed at pH 5, but not at pH 7.6. Owing to the magnitude of the inhibition constant, product inhibition was not observed in normal turnover hydrolysis experiments. Inhibition by the phenol product of hydrolysis was not observed at concentrations ranging between 0.1 and 1.0 mM for the substrates 2-chloro-4-nitrophenyl sulfate, 2-carboxyphenyl sulfate, and 2-nitrophenyl sulfate. Similarly the catalyzed hydrolysis of 2-chloro-4-nitrophenyl sulfate was unperturbed in the presence of phenol products from other substrates, e.g., phenol (100 mM), 2,4-dinitrophenol (0.2 mM), and salicylic acid (1 .O mM) . Inhibition of the enzyme catalyzed hydrolysis of 2-chloro-4-nitrophenyl sulfate by 4-carboxyphenyl sulfate under conditions where change in the concentration of either ester is negligible also proved to be of the linear competitive type with Ki = 8.3 x 1O-4 M. This value should be compared with K, = 7.5 x 10m4 M determined for 4-carboxyphenyl sulfate under identical conditions. Inhibitory effects for various aliphatic and aromatic sulfate and phosphate esters on the enzyme catalyzed hydrolysis of p-NPS are shown in Table II. Values for the inhibition constants, K,, were determined at pH 4.04 and 7.60. The inhibition exhibited by estrone-3-sulfate, N-CbzL-Phe-L-Tyr-O-sulfate methyl ester, 4nitrophenyl phosphate, and phenolphthalein disulfate was competitive at pH 4.04. Similarly at pH 7.60, the inhibition shown by dodecyl-l-sulfate, 4-nitrophenyl phosphate, and phenolphthalein disulfate was competitive while indoxyl sulfate revealed a mixed type of inhibition. Throughout the tenfold range of inhibitor concentrations employed, linear Dixon plots were obtained. Graphs of log (u,/u - 1) against log

ET AL.

Fm. 4. Product inhibition of arylsulfate sulfohydrolase II shown by a Dixon plot of l/u for the hydrolysis of the variable substrate 2-chloro-4nitrophenyl sulfate against inorganic sulfate concentration at pH 3.97, 37.5”C. TABLE

II

INHIBITION OF ARYLSULFATE SULFOHYDROLASE II BY VARIOUS SULFATE AND PHOSPHATE ESTERS

(37.5”C. I = 0.3)” Ester Hexyl-l-sulfate Decyl-l-sulfate Dodecyl-l-sulfate o-Galactose-6-sulfate n-Glucose-6-sulfate n-Glucose-l-phosphate Androstenolone-3@-sulfate Estrone-3-sulfate’ Indoxyl sulfate L-Tyrosine-O-sulfate N-Cbz-L-Phe-L-Tyr-o-sulfate methyl ester’ 4-Nitrophenyl phosphated Phenolphthalein disulfate’

K, x 10’ (pH 4.04)

K, x lo2 (pH 7.60)

b

0.725 -

13.3

25.3 -

13.7 35.5 0.13

2.00 2.12

’ A dash indicates that no inhibition was observed. b Nonlinear inhibition was found owing to dissociation of the enzyme.

c K, 2 K,. d K, determined at pH 4.29 and 6.0, respectively. p K, based on M of phenolphthalein disulfate.

i for the competitive inhibitors at pH 4.04 and 7.60 yielded straight lines with a slope of 1 implying the absence of any cooperative interaction between subunits. The competitive aromatic sulfate esters

PROPERTIES

OF ARYLSULFATE

were then tested as possible substrates. The ratio of V for estrone-3-sulfate and N-Cbz-L-Phe-L-Tyr-O-sulfate methyl ester to p-NPS are 1.05 and 0.058, respectively, under the conditions noted in Table II. Values of K, are 3.44 + 0.79 x 10m4 M and 10.5 & 3.63 x lo-” M, respectively. Trapping studies. The results of the I80 tracer experiments designed to demonstrate a possible reversible pathway between inorganic sulfate and a sulfuryl enzyme intermediate are given in Table III. The data show no apparent catalyzed exchange of oxygen atoms occurs between the solvent and inorganic sulfate even at a twenty fold greater enzyme concentration and a much longer incubation time than normal kinetic experiments. The control experiments reveal that one atom of solvent is incorporated per molecule of inorganic sulfate during both the spontaneous and enzymic hydrolysis of p-NPS. It appeared plausible that a redox mechanism might operate in some reactions catalyzed by arylsulfate sulfohydrolase since the substrates are primarily aromatic and sulfite is a potent competitive inhibitor. A pathway proceeding through

4 that features an enzyme bound sulfite and unsaturated ketone might be amenable to interception with DTNB or sodium borohydride. Incubation of sodium borohydride and 2-chloro-4-nitrophenyl sulfate with enzyme followed by dialysis led to recovery of enzyme with unchanged sp act. In turnover experiments with DTNB present, no sulfite formation was observed. DISCUSSION

Physical properties. A previous investigation of arylsulfate sulfohydrolase II revealed that the enzyme may possess two active sites per molecule (5). This postulate stemmed from titration experiments in which the enzyme was treated with onitrophenyl oxalate, an irreversible inhibitor of the enzyme. The completely inactivated protein which resulted from the ti-

379

A. ORYZAE TABLE

III

I80 TRACER STUDIES WITH ARYLSULFATE SULFOHYDROLASE II Sample’

(I) (II) (III) (IV)

Enzyme + Na,SO, Enzyme + p-NPS Na,SO, p-NPS

Atom% ‘80 enriched solvent”

4.3 4.3 4.3 5.0

Atom B I80 found in inorganic sulfaw

Atoms of solvent incorporated Per molecule of inorganic sulfate’

0.0

0.0

1.09

0.9

0.0

0.0 1.2

1.7

u For conditions see Experimental. b Aqueous acetate buffer, pH 4.0. c Error bounds * 10%. These values were corrected for the natural abundance of I80 found in the control, Sample III.

tration had a 2.2:1 oxalate ester:enzyme stoichiometry. Previous attempts to carry out the gel electrophoresis of arylsulfate sulfohydrolase II at pH 4.3 and 7.5 according to the procedure of Williams et al. were unsuccessful (23). Migration of the enzyme was not observed at pH 4.3 while the slow migration at pH 7.5 often resulted in weak diffuse bands. Consequently, any study of the electrophoretic behavior of the purified enzyme under varying pretreatment conditions had to be conducted at pH 8.6 with the assumption that any effect of pH and dissociating agents would be essentially irreversible. Under these conditions, a single band was obtained after the enzyme had been exhaustively dialyzed in acetate buffer at pH 4.04. After incubation of the enzyme in 0.4% dodecyl sulfate or 8 M urea at pH 4.04, two distinct bands were found. Plausible rationales for this behavior in the presence of these reagents include: (1) the separation of inactive aggregated material from the enzyme; (2) a partial dissociation of the native enzyme into two or more identical subunits; or (3) a total dissociation of the native enzyme into two distinct subunits of differing molecular weights and electrophoretic mobilities. To distinguish between these possibili-

380

SAMPSON

ties, equilibrium sedimentation experiments were carried out at the indicated pretreatment conditions. At pH 4.04 in the presence of 8 M urea and at pH 4.83 apparent molecular weights of 53,200 i 2400 and 94,900 * 7100 were determined, respectively. The relationship between these two values suggests that the enzyme is composed of two identical subunits. The nonlinearity noted at pH 4.04 (Fig. 1) is in accord with a nonuniform distribution of species throughout the cell, indicating the enzyme is only partially dissociated under these conditions. It is probable that a similar explanation applies to the behavior observed with gel electrophoresis. The nonlinear inhibition observed with dodecyl sulfate at pH 4.04 further supports this contention. Data collected at pH 7.60 would suggest that the enzyme does not dissociate appreciably into subunits at this pH. The electrophoresis of the enzyme after dialysis and again after pretreatment with 8 M urea or 0.4% dodecyl sulfate revealed only one stationary band. The molecular weight determined from equilibrium sedimentation experiments was 91,300 * 4700, although nonlinear plots of In C against X2 were obtained in the presence of 8 M urea. Finally, dodecyl sulfate was shown to be a competitive inhibitor of the enzymic hydrolysis of p-NPS at pH 7.60. It is likely that the high negatively charged state of the protein at this pH may prevent the binding of dodecyl sulfate at sites other than the active site. The effect of dodecyl sulfate on arylsulfohydrolase A from ox liver has also been shown to be governed by the net charge on the molecule (isoelectric point = 3.6) (24). Arylsulfohydrolase A from ox liver exists as a monomer unit at pH 7.5 with a molecular weight of 107,000 and as a tetramer at pH 5.0 with a molecular weight of 411,090. At the relevant pH, dodecyl sulfate dissociates the tetramer but not the monomer unit into smaller identical subunits of molecular weight, 24,000. It is unfortunate that further studies of the concentration dependent equilibrium process are hampered by unavailability of sufficient nure enzvme. However. the re-

ET AL.

vised molecular weight is close to that reported by Rasburn et al. for an isoenzyme of arylsulfohydrolase from A. orytae (25). Although neither Drnec nor Rasburn et al. report subunit structure for the protein, it is apparent that this possibility should be weighed in future studies on physical properties of the protein. Based on the subunit data presented above, it is quite likely that below pH 4.0, the decrease in V may correspond to dissociation of the enzyme-substrate complex into inactive or only partially active subunits. However, there is presently no evidence to support the formation of subunits on the basic side (pH 7.60). The similarity between the values of pK,,,, in the enzyme-substrate complex and pK,,, in the free enzyme suggests the same functional group may be involved in both instances. Furthermore, it should be stressed that with the exception of the irreversible inhibition by p-nitrophenyl oxalate, there is no kinetic demonstration for the involvement of both subunits in the catalytic process. Data for evaluation of K, or K, show no deviation from behavior expected for a single site enzyme. It is quite possible that the ca. loo-fold variation in substrate concentration was insufficient to disclose catalytic activity at individual subunits, without invoking alternate hypotheses. Mechanism. A main objective of the present investigation was to seek support for a reaction sequence involving a covalent sulfuryl enzyme species represented as ES’ in equation (5). The pH-dependence of the kinetic parameters would be satisfied E+S

K,

ES +P,

k, g

ES’ 2

E+P,

(5)

by including additional E and ES species as in (1) or alternatively by a pH dependent change in the identity of the rate-controlling step. The chief support for this reaction sequence is based on the insensitivity of V to the pK, of the departing phenol (p = 0 at pH 4 and 7.6) in accord with the k, sten being largelv rate deter-

PROPERTIES

OF ARYLSULFATE

A. ORYZAE

381

mining (3). The near identity of the /3 change of I80 from solvent into inorganic sulfate via steps 12, and k-, was not values suggest that if a change in the rate-limiting step accompanies a change in achieved. The incorporation of one atom of I80 per molecule of inorganic sulfate durthe pH, it is not manifest in the structureing the catalyzed hydrolysis is in agreereactivity correlations. ment with a mechanism featuring S-O At pH 4.0, inorganic sulfate is a linear bond cleavage and apparently is a common competitive inhibitor of the enzymic hymechanistic feature for a variety of aryl drolysis of Z-chloro-4-nitrophenyl sulfate consistent with the above scheme in which sulfatases (29). A kinetic test of reaction sequence (5) sulfate is the last released product or one in also was executed. It can readily be shown which both sulfate and phenol are released simultaneously (26). Differentiation be- that for this scheme tween these two postulated reaction K, = K,k, (6) schemes could be obtained by determining k, + k, the type of inhibition exhibited by the phenol product (26). Unfortunately inhibiAccording to equation (6) K, > KS, if k, > tion by 2-chloro-4-nitrophenol is not ob- k, which is the precondition for rate-determining breakdown of ES’ (30). The actual served at the highest concentration practidissociation constant of an enzyme-subcable to use in the spectrophotometer. Similarly, the catalyzed hydrolysis of 2- strate complex, KS, was evaluated indirectly by determining the dissociation chloro-4-nitrophenyl sulfate is not inhibconstant, K,, of an enzyme-inhibitor comited by the phenol product of other subplex in a situation where a poor substrate strates even at concentrations as great as acts as a linear competitive inhibitor. The 100 mM. In an analogous study, Roy and ester, 4-carboxyphenyl sulfate, was emNichols have shown that inorganic sulfate ployed both as a substrate for the enzyme behaves as a linear competitive inhibitor of as well as an inhibitor of the sulfohydrolase arylsulfatase A from ox liver, whereas no catalyzed hydrolysis of 2-chloro-4-nitroinhibition by the phenol product is de- phenyl sulfate. The value of K, x K, within experimental error. Consequently, tected (27). With the latter enzyme, sulfate the condition K, > KS is not observed. also acts as an activator of the modified Values of K, x Ki were also found for two enzyme form produced during the catalytic sulfates listed in Table II. cycle. However, the enzyme from A. orytae In view of the collective results, it is exhibits none of the anomalous kinetics concluded that the rate-limiting step in the found with the ox liver protein associated hydrolysis of sulfate esters by arylsulfate with sulfate activation (27). sulfohydrolase II is not the breakdown of a More conclusive support for the accumusulfuryl enzyme intermediate. An alternalation of a transient ES’ species was sought tive mechanism including rate-determinvia three mechanistic probes. Direct detecing conformational changes or product deabsorption cannot be excluded. Howtion of the intermediate employing the ever, no induction period was noted in stopped-flow technique at pH values spanstopped-flow experiments that would ning the V-pH profile was unsuccessful. imply a transformation from an inactive to Secondly partitioning of a sulfuryl enzyme a catalytically active enzyme form. It is intermediate between added amine nucleoequally plausible that the enzyme might philes and water should lead to an increase level the substituent effects of the aromatic in Vowing to the increased rate of desulfamoiety on the hydrolysis rates. In this tion since the nonenzymic hydrolysis of manner one may retain scheme (5) with k, p-NPS is catalyzed by a series of amine rate-limiting or bypass ES’. The implicanucleophiles (28). No increase in V was tions of this possibility are of interest since observed. Thirdly, enzyme catalyzed ex- the finding of identical rates of reaction for

382

SAMPSON

different substrates is frequently used as evidence for the existence of a covalent substrate-enzyme intermediate. A quantitative estimate of the magnitude of this leveling effect may be obtained from the structure-reactivity correlation for the nonenzymic hydrolysis of sulfate monoesters. The small fl value observed for the specific acid catalyzed hydrolysis of these compounds, fl = -0.2, over a wide range of substituent effects implies only a small dependence upon the pK, of the departing group (31). Therefore, the enzyme has only to reduce the effect of the substituent by ca. 10 fold compared to the nonenzymic hydrolysis in order to cause the observed results, provided a similar mechanism obtains. Substrate specificity. The compounds in Table II were examined in order to more accurately define the structural requirements for arylsulfate sulfohydrolase II. Since the hydrolysis reaction for many of the esters could not be monitored spectrophotometrically, their potential as substrates was determined by their inhibitory effect on the enzyme catalyzed hydrolysis of p-NPS. Perhaps the most interesting observation is that a polypeptide of tyrosine-O-sulfate binds remarkably well to the enzyme, whereas the unesterified compound acts as neither substrate nor inhibitor. Apparently the active site does not readily accommodate the mainly zwitterionic tyrosine-O-sulfate. Indoxyl sulfate, which is not an inhibitor at pH 4.0, gives a mixed type of inhibition at pH 7.6. Assuming an approximate pK, of 4.9 for the ring nitrogen (32), indoxyl sulfate should be mainly in the protonated form at pH 4.0 and accordingly possesses a low affinity for the enzyme. This conclusion includes the observations of Boyland et al. that oaminoacyl sulfates bearing amino groups protonated under the assay conditions were not hydrolyzed by the A. oryzae enzyme (33). Collectively the remainder of the data in Table II implicates the necessity for an aromatic moiety on the substrate. With the exception of dodecyl sulfate, the aliphatic and monosaccharide sulfate and -phosphate esters do not appear to bind to the

ET AL.

enzyme. Similarly, estrone-3-sulfate acts as a substrate or inhibitor, a finding that led to the design of the peptide substrate. On the other hand, androstenolone-3@sulfate, a nonaromatic steroidal ester, reveals no detectable inhibition. Thus substrate activity apparently requires an aromatic sulfate ester whose binding to the enzyme is enhanced by incorporating the aromatic ring in a hydrophobic backbone but is decreased by the presence of positively charged substituents. Finally, it is of interest that V for the polypeptide sulfate deviates by an order of magnitude from that predicted from the earlier structure-reactivity correlation derived for a series of phenyl esters with highly polar substituents. The enzyme catalyzed hydrolysis of estrone-3-sulfate and the polypeptide sulfate imply the probable existence of a second class of compounds that incorporates a common structural feature of a hydrophobic backbone substituted para to the sulfate moiety. This group may well include the actual physiological substrate. REFERENCES 1. CHERAYIL, J. D., AND VAN KLEY, H. (1963) Fed. Proc. 22, 241. 2. DRNEC, J. F. (1968) Ph.D. Dissertation, St. Louis University, St. Louis, Missouri. 3. BENKOVIC, S. J., VERGARA,E. V., AND HEVEY, R. C. (1971) J. Biol. Chem. 246, 4926. 4. CHERAYIL, J. D. (1963) Ph.D. Dissertation, St. Louis University, St. Louis, Missouri. 5. BENKOVIC, S. J., AND FEDOR, J. M. (1972) J. Amer.

Chem. Sot. 94, 8928. 6. BURMA, D. P. (1953) Anal. Chem. 25, 549. 7. BARTON, G. M., EVANS, R. S., AND GARDNER, J. A. F. (1952) Nature (London) 170, 250. 8. DODGSON, K. S., ROSE, F. A., AND TUDBALL, N. (1959) Biochem. J. 71, 10. 9. GREENSTEIN,J. P., AND WINITZ, M. (1961) Chemistry of Amino Acids, Vol. I, John Wiley and Sons, New York. 10. WOODWARD,R. B., OLOFSON,R. A., ANDMAYER, H. (1961) J. Amer. Chem. Sot. 83, 1010. 11. BLOCK, R. J., DURRUM, E. L., ANDZWEIG, G. (1958) A Manual of Paper Chromatography and Paper Electrophoresis, Academic Press, New York. 12. REITZ, H. C., FERREL, R. E., OLCOTT, H. S., AND CONRAT-FRAENKEL, H. (1946) J. Amer. Chem. Sot. 68, 1031. 13. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L..

PROPERTIES

14. 15. 16. 17.

18. 19. 20.

21. 22. 23.

OF ARYLSULFATE

AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. HUGGINS, C., AND SMITH D. E. (1947) J. Biol. Chem. 170, 391. ORNSTEIN, L., AND DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 321. CHRAMBACH, A., REISFELD, R. A., WYCKOFF, M., AND ZACCARI,J. (1967) Anal. Biochem. 20,150. CHERVENKA, C. H. (1969) A Manual of Methods for the Analytical Ultracentrifuge, Spinco Division of Beckman Instruments, Palo Alto, California. CLELAND, W. W. (1969) Aduan. Enzymol. 29, 1. BOYER, P. D., GRAVES, D. J., SUELTER, C. H., AND DEMSEY, M. E. (1961) Anal. Chem. 33,1906. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70; TORII, K., AND BANDURSKI, R. S. (1967) Biochim. Biophys. Acta 136,286. SCHACHMAN, H. K., AND EDELSTEIN, S. J. (1966) Biochemistry 5, 2681. ALBERTY, R. A., AND MASSAY, V. (1954) Biochim. Biophys. Acta 13, 347. WILLIAMS, D. E., AND REISFELD, R. A. (1964) Ann.

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N. Y. Acad. Sci. 121, 373. 24. NICHOL, L. W., AND ROY, A. B. (1966) Biochemistry 5, 1379. 25. RASBURN, M., AND WYNN, C. H. (1973) Biochim. Biophys. Acta 293, 191. 26. CLELAND, W. W. (1963) Biochim. Biophys. Acta 67, 188. 27. NICHOLLS, R. G., AND ROY, A. B. (1971) Biochim. Biophys. Acta 242, 141. 28. BENKOVIC, S. J., AND BENKOVIC, P. A. (1966) J. Amer. Chem. Sot. 88, 5504. 29. SPENCER, B. (1958) Proc. Intern. Symposium Enzyme Chem. Tokyo and Kyoto, 2, 96. 30. GUTFREUND, H., AND STURTEVANT, J. M. (1956)

Biochem. J. 63, 656. 31. BENKOVIC S. J., AND DUNIKOSKI, L. K. (1970) Biochemistry 9, 1390. 32. PERRIN, D. D. (1965) Dissociation Constants of Organic Bases in Aqueous Solutions, Butterworth, London. 33. BOYLAND, E., MANSON, D., SIMS, P., AND WILLIAMS, D. C. (1956) Biochem. J. 62, 68.