THE JOURNAL OF Bmm~rcar, CHEMISTRY Vol. 246, No. 16, Issue of August 25, PP. 492G4933, 1971

Printed in U.S.A.

Purification and Properties of an Arylsulfatase

from Aspergihs oryz;ae*

(Received for publication, January 25, 1971)

STEPHEN J. BENKOVIC,$ E. V. VERGARA, AND R. C. HEVEY~

From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

SUMMARY

Arylsulfate sulfohydrolase II (EC 3.1.6.1) has been purified from Aspergillus oryzae. The purified arylsulfatase appears to be homogeneous on the basis of disc gel elec- trophoresis, sucrose density gradient centrifugation, and equilibrium ultracentrifugation. Physical properties and amino acid composition have been determined indicating the enzyme to have a molecular weight of 65,000 f 2,000 with a high content of aspartic and glutamic acid.

Kinetic constants for the hydrolysis of a series of substi- tuted phenyl sulfate esters at pH 4.0 and 7.5 have been determined. Plots of log V,,,/K, and log V,,, against pKa show that, as the electron-withdrawing ability of the substituent increases, log V,,,f K, increases with decreasing pK,, while log V,,, is independent of pK,. These results are interpreted in terms of a covalent sulfuryl enzyme inter- mediate or a rate-determining conformational change.

The arylsulfate sulfohydrolases (EC 3.1.6.1)) which catalyze the hydrolysis of arylsulfates, have been detected in most animal tissues, in many microorganisms, and in the seeds of a few species of higher plants (1). Such a widespread distribution would imply a rather fundamental function for these enzymes; however, their role has not been fully understood up to the present.

An insight into the catalytic groups at the active site has been provided by the specificity studies on the arylsulfatase from Alcaligenes metalcaligenes (2) and by the action of group-specific protein reagents on arylsulfatase A from ox liver (3). Dodgson, Spencer, and Williams (2) demonstrated a relationship between the logarithm of the rates (I’,,,) of hydrolysis and Michaelis constants (Km) for a series of substituted aromatic sulfates and the Hammett substituent constant (u). This result paralleled the observed (I dependence of the rates for specific acid hydrolysis of the same series of esters, leading the authors to conclude that decomposition of the enzyme-substrate complex involved specific

* This research was supported by Public Health Service Grant GM-13306.

$ National Institutes of Health Career Development Awardee, Alfred P. Sloan Fellow, 1968 to 1970.

$ National Institutes of Health Predoctoral Fellow, 1967 to 1971.

acid catalysis. Evidence for the possible involvement of tyrosyl residues at the active site has been derived from the inactivation of arylsulfatase A by treatment with tetranitromethane or N-acetylimidazole. However, much of the proposed mechanism of the enzyme-catalyzed reaction is still founded on results ob- tained in model systems (4).

One major difficulty has been the unavailability of a suffi- ciently homogeneous preparation which might be suitable for chemical investigations. This report presents a scheme for the purification of arylsulfate sulfohydrolase II from Aspergillus oryzue under the cultural conditions described by Cherayil (5) and discusses to some extent its physical, catalytic, and chemical properties.

EXPERIMENTAL PROCEDURE

Materials

A culture of A. oryzae, NRRL-449 was kindly donated by Dr. J. J. Ellis of the Agricultural Research Service of the United States Department of Agriculture. Bacto-malt agar was ob- tained from Difco Laboratories. Generous supplies of wheat bran were made available by Agway, Inc.

Bio-Rex 70 was obtained from Bio-Rad Laboratories; Sepha- dex G-200 and blue dextran 2000 were from Pharmacia; poly- acrylamide gel electrophoresis reagents were from Distillation Products, Inc.; Coomassie brilliant blue R250 was from Colab Laboratory, Inc.; crystalline bovine serum albumin, p-nitro- phenyl phosphate, p-nitrophenyl-, and 5-nitrocatechol sulfates were from Sigma. 2-Nitro-, 3-nitro-, 2,4-dinitro- and 2-chloro- 4-nitro sulfates were prepared and purified by the modified procedure of Burkhardt and Wood (6) as described by Fendler and Fendler (7). The dipotassium salt of salicyl sulfate was prepared and purified according to the procedure of Benkovic (8). The potassium salts of standard solutions were assayed spectrophotometrically after complete acidic hydrolysis and were shown to be at least 99% pure. Spectral data for phenols and sulfate esters are listed in Table I. All phenols, except 2-chloro- 4-nitrophenol and salicylic acid which were recrystallized, were vacuum sublimed prior to use.

Protein Determination

Protein concentrations, unless otherwise stated, were deter- mined by the method of Lowry et al. (9), with crystalline bovine serum albumin as a standard.

4926

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Issue of August 25, 1971 8. J. Benkovic, E. V. Vergara, and R. C. Hevey 4927

Enzyme Assay

Arylsulfate sulfohydrolase activity was assayed by a slight modification of the method of Huggins and Smith (10). The reaction mixture consisted of 1500 pmoles of sodium acetate buffer (0.5 M, pH 5.8), 5.0 pmoles of p-nitrophenyl sulfate, and 1.0 ml of the enzyme preparation in a total volume of 5.0 ml. After incubation for 1 hour, at 37.5”, the reaction was stopped by the addition of 5.0 ml of 1.0 M sodium hydroxide and the liberated p-nitrophenol was measured in a Beckman DU-2 spectrophotom- eter at 420 mp against the appropriate controls. A molar absorp- tivity of 14,600 at 420 rnp was used in the calculations.

Similar assays were done in sodium acetate buffer, pH 3.9, and the ratio of activity at pH 5.8 to that at pH 3.9 was calcu- lated (5). The catalytic reaction was linear with the enzyme concentration used in the assay.

One unit of arylsulfate sulfohydrolase activity is defined as the amount of enzyme required to produce 1 pmole of p-nitro- phenol per min at 37.5” under the described assay conditions. The specific activity is expressed as units of activity per mg of protein.

Kinetics

The hydrolysis of the substituted phenyl esters was followed spectrophotometrically with a Gilford 240 thermostated spec- trophotometer at an appropriate wave length (Table I). In a typical determination 0.050 ml of enzyme stock solution was added to 0.35 ml of buffer in a l-cm cell and placed in the ther- mostated cell compartment of the spectrophotometer which was maintained at 37.5” (hO.1”) with a Haake circulating water bath. The buffers utilized were sodium acetate (0.2 M, M = 0.2), Tris acid maleate-sodium hydroxide (0.2 M, p = 0.2), and Tris hydro- chloride (0.2 M, p = 0.2). After the temperature had equili- brated (10 min), substrate (0.10 ml, previously equilibrated to 37.5” in an immersion circulating water bath) was added and absorbance readings were taken every 30 sec. Initial velocities were determined from the slopes of straight lines obtained when absorbance was plotted against time after correction for non- enzymic hydrolysis of the substrate. No product or pH inhibi- tion was detected. The pI1 of the reaction solution at the end of the run was determined to &0.02 pH unit on a Radiometer model 22 pH meter with a model pH 630 Pa scale expander and a Metrohm type XEA125 combination electrode or a Radiometer G.K.2021 B Electrode. The experiments were repeated at different substrate concentrations made up from a stock sub- strate solution which was periodically analyzed for any ester decomposition. In the case of 2,4-dinitrophenyl sulfate the solutions were made up immediately prior to use owing to the rapid hydrolytic decomposition of this substrate.

The apparent K, of the enzyme was determined by using both purified and partially purified enzyme (end of Steps 3 and 4 in preparation). Both preparations gave the same apparent Km for 2nitrophenyl sulfate, 4-nitrophenyl sulfate, and salicyl sulfate. For the buffers utilized, enzymic activity was found to be independent of buffer concentration and ionic strength up to 0.4 M. Since there is no direct method for measuring the active site concentration of arylsulfatase in solution, a relative activity measurement of the enzyme stock solution was used (pH 3.9) and corrections were applied for any enzyme activity lost. All kinetic data were subjected to statistical analysis and their standard error was evaluated by using the program (HYPER)

TABLE I Spectral data for phenols and sulfate esters

All measurements made at 37.5”, J.L = 0.2.

Sulfate ester

4-Nitrophenyl

2-Nitrophenyl

3-Nitrophenyl

2,4-Dinitrophenyl 5-Nitrocatechol

I-Chloro-4-nitro- phenyl

Salicyl

a In 0.2 M HCl. b In 0.2 M KOH.

-7

WC%%? length

nm

400 350d 416 351 392 333 360 515 345 402 316 302.5

Phenol C’

5,280

2,990 201

1,960 2,180

6,850~

8,360 3,720"

Phenolate cb

18,300

4,720 1,230 1,530 1,080

14,800 11,200c

17,100 1,360

-

__

-

Ester 8

521

439 19

520 106

5,030

2,950 83

c Determined at pH 4.02 in 0.2 M sodium acetate buffer, p = 0.2 with NaCl.

d Isosbestic point of p-nitrophenol.

described by Cleland (11) which is based on a weighted fit to the reciprocal form of the Michaelis-Menten equation (12). A minimum of five substrate concentrations was used for each analysis.

Inhibition studies were conducted with a reaction mixture which consisted of 30 pmoles of NaOAcl (0.05 M) buffer (pH 4.8), 1.0 pmole of p-NPS, 0.2 ml of inhibitor (0.005 M), and 0.2 ml of the purified enzyme in a total volume of 1.2 ml. After incubation at 37.5” for 30 min, the reaction was stopped by the addition of 1.0 ml of 1.0 M NaOH. Initial velocities were calculated from the amount of p-nitrophenol liberated in a 30-min incubation period. All solutions were prepared in 0.05 M NaOAc buffer and the pH was adjusted to 4.8.

Growth Conditions

A. oryzae strain NRRL-449 was grown on malt-agar slants at room temperature. A culture medium (5) consisting of 200 g of wheat bran, 3.0 g of urea, 6.5 g of diammonium phosphate, and 70.0 ml of distilled water was autoclaved, cooled, and inocu- lated with a spore suspension of the mold from the malt-agar slants. Growth was allowed to take place at room temperature until the wheat bran was completely covered with the mold mycelium. The extraction of the mold bran and precipitation of the active protein with acetone were by the method of Cherayil.

Purijication Procedure

All operations were performed at O-5’ unless otherwise stated. Step 1: Extraction of Acetone-dried Powder-Fifteen grams of

the acetone-dried powder were stirred in 75 ml of ice-cold 0.03 M

NaOAc buffer, pH 4.8. The extract was centrifuged at 1800 rpm for 20 min, and the residue was re-extracted with 38 ml of buffer. The extracts were combined; the pH was adjusted to 4.8 with 1.0 M acetic acid, and centrifuged further to remove any insoluble material.

1 The abbreviations used are: NaOAc, sodium acetate; p-NPS, p-nitrophenyl sulfate; p-NPP, p-nitrophenyl phosphate.

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4928 Arylsul$atase from A. oryxae : Purification and Properties

Step 2: Chromatography on Bio-Rex 70-The clear extract from Step 1 was added to a Bio-Rex 70 column (3 x 36 cm) previously equilibrated with 0.03 M NaOAc buffer, pH 4.8. The column was washed with the same buffer; elution was continued with a linear gradient consisting of 600 ml of 0.03 M NaOAc buffer, pH 4.8, and 600 ml of 0.5 M Tris-acetate buffer, pH 7.5, and com- pleted with an additional 300 ml of the Tris-acetate buffer. Fractions of 10 ml were collected at a flow rate of about 1.0 ml per min. The fractions containing arylsulfate sulfohydrolase II (see “Discussion”) were pooled, concentrated to approximately 10 ml by ultrafiltration with a Dia-Flo UM-10 membrane (Amicon Corporation), and dialyzed against 0.03 M NaOAc buffer, pH 4.8.

Step 3: Chromatography on Sephadex G-200-The dialyzed sample was applied to a Sephadex G-200 column (4 x 92 cm) which was previously equilibrated with 0.03 M NaOAc buffer, pH 4.8. The enzyme was eluted m-ith the same buffer. Frac- tions of 5 ml were collected at a flow rate of 0.17 ml per min. The fractions with the highest activity were combined, con- centrated by ultrafiltration, and dialyzed against 0.05 M Tris- glycine buffer, pH 8.9.

Step 4: Preparative Gel Electrophoresis-The enzyme was fur- ther purified by preparative polyacrylamide gel electrophoresis (13) on the Poly-Prep 200 (Buchler Instruments, Inc.). The Tris-glycine gel system recommended by Buchler for the Poly- Prep (14) was adopted except that the concentrating gel was chemically polymerized with ammonium persulfate in Tris- chloride buffer, pH 6.7. Electrophoresis was carried out at 5” with a constant current of 50 ma. Fractions of 10 ml were collected at a flow rate of 1.0 ml per min, with 0.1 M Tris-chloride, pH 8.1, as the elution buffer. The fractions which were shown to be homogeneous by analytical gel electrophoresis were pooled, concentrated by ultrafiltration, and dialyzed against 0.03 M

NaOAc buffer, pH 4.8. This enzyme was used for all further studies.

Analytical Gel Electrophoresis

Polyacrylamide disc gel electrophoresis of the enzyme was carried out at pH 8.3 as described by Ornstein and Davis (15)

TABLE II

Purifcalion of arylsulfafe sulfohydrolase IIfrom Aspergillus oryzae

step

1. Extraction of the acetone- dried powder.. .

2. Chromatography on Bio-Rex 70, . . . .

3. Chromatography on Sepha- dex G-200.. . .

4. Preparative disc electro- phoresisb. c.. . . . .

a Enzyme assays at pH 5.8.

Total enzyme Total unitsa protein

97.7 382

43.3 45.5

15.1 11.8

Vol. 246, No. 16

and at pH 4.5 as suggested by Williams and Reisfield (16). Gels were fixed and stained for protein with Coomassie brilliant blue (17) and for enzyme activity with 0.005 M p-NPS.

sucrose Density Gradient Centrijugation

Gradients of 5 to 25% sucrose in 0.03 M NaOAc buffer, pH 4.8, were prepared by the method of Martin and Ames (18) as modified by Pazur, Kleppe, and Anderson (19). The enzyme, 0.280 mg in 0.2 ml of the same buffer, was layered on the top of one gradient and centrifuged at 55,000 rpm for 12 hours in the SW 65 L rotor of a Beckman L2-65 ultracentrifuge. With a density gradient fractionator (ISCO model D), 24 fractions were collected and then assayed for arylsulfate sulfohydrolase activity.

Ultracenlrijugation

Sedimentation equilibrium ultracentrifugation was performed in the Beckman model E analytical ultracentrifuge with an ultraviolet scanner operated at 280 mp. A Yphantis multi- chamlel equilibrium centerpiece and two Kel-F double sector cells were used in the An-D rotor. The enzyme, at a concentra- tion of 0.4 mg per ml at 280 rnp, was centrifuged at 5” and 14,000 rpm for 20 hours in 0.03 1~. NaOhc buffer, pH 4.8.

Actual calculations on the raw data obtained from the scanner traces were executed on the IBM 360/67 computer, with a FORTRAN IV program (provided by Mr. J. M. Schorr of the Department of Biochemistry). T$y using a least squares regres- sion analysis, the slope of the Yphant,is plot of In C against X2 (20) and the standard error of the estimate for the slope were computed. The apparent molecular weight of the enzyme was calculated from this slope.

Amino Acid Analysis

Three milligrams of the enzyme were dialyzed exhaustively against distilled water and then hydrolyzed for 24 hours with constant boiling 6 N WC1 at 110”. Duplicate samples of the hydrolgsate were analyzed on :t Technicon automatic amino acid analyzer (21).

RESULTS

Pur$cation-Table II summarizes the purification of aryl- sulfate sulfohydrolase II from A. oryzae. Although preparative disc electrophoresis increased the specific activity of the enzyme

Specific only slightly, it was most effective in removing the two remaining activity Yield

protein contaminants in the enzyme preparation. The over-all -- purification was 26.fold with a 7.8% yield. This represented a mn&T/mg % purification of 762-fold based on the specific activity of the

0.048 100 homogenized wheat bran.*

Homogeneity-The enzyme appeared to be homogeneous on the

0.256 50.5 basis of disc gel electrophoresis, sucrose density gradient cen- trifugation, and ultracentrifugation.

0.952 22.9 Disc gel electrophoresis of the enzyme at the various steps in the purification is shown in Fig. 1. After preparative disc

1.27d 7.8 electrophoresis, the enzyme migrated as a single protein band which was shown to be enzyme active by staining with p-NPS.

Centrifugation of the purified enzyme in a sucrose gradient at b Enzyme and protein assays were done on the dialyzed sample.

Tris and glycine, the buffer components of the gel system, have pH 4.8 revealed a single, symmetrical, protein peak which exactly

been reported (22) to interfere with the method of Lowry et al. corresponded to the enzyme activity peak.

(9). Equilibrium sedimentation studies gave results that were con-

e The enzyme had an activity ratio of 1.1 after this step. * This was based on an average specific activity of 0.002 of the d This may be compared to a value of 10.9 reported by Drnec mold bran extracts. Variation in the specific activity was ob-

(28). The reason for this discrepancy is not known. served from one batch to another.

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FIG. 1. Disc gel electrophoresis at various steps in the purifica- tion. From left to riaht: Ster, 1. Sten 2. Sten 3, and the purified arylsulfate suifohydrolase Ii (60 pgj after preparative &sc gel electrophoresis.

sistent with a homogeneous preparation. A linear plot of In C against X2 is seen in Fig. 2.

Physical Properties-From the slope of the linear plot of In C against X2 and a partial specific volume of 0.722 determined from the amino acid analysis (23), a molecular weight of 65,000 =t 2,000 was calculated from duplicate determinations.

The amino acid composition of the arylsulfate sulfohydrolase is presented in Table III. Average micromoles were calculated by comparison with standard amino acids, and these were nor- malized with respect to phenylalanine. The number of residues per molecule was calculated from the molecular weights of the amino acids and an enzyme molecular weight of 65,000. Aspartic acid, glycine, and glutamic acid were the most frequently oc- curring amino acids, while cyatine, histidine, methionine, and arginine were present in the lowest amounts.

Catalytic Properties-The pH optimum of the purified enzyme was determined over a pH range of 3.6 to 8.8. The enzyme was active in the pH range 4.0 to 6.0 with optimal activity at pH 4.8. The activity ratio of pH 5.8 to pH 3.8 is 1.1.

The occurrence of irreversible destruction of the enzyme on both sides of the pH optimum was tested initially by exposing the enzyme to a range of pH values (24 hours, 5”) and then assaying the activity after readjusting the pH to its optimum value. In- activation on the alkaline side of the pH rate profile is fully reversible, whereas inactivation on the acidic side is only partially reversible. Further experiments over 72 hours at 4” (pH 3.6 to 8.8) indicated a 94% recovery of activity at pH 4.8. Over the same period of time, 75% of the activity was recovered at pH 4.5, 46% at pH 4.0, and 430/b at pH 3.6. The enzyme, at its optimum pH, was found to be stable in the frozen state over 1 month. Incubation studies at t~he reaction temperature of 37.5” showed that the enzyme was stable for 30 min, losing 1% of its activity after 50 min and 2% of its activity after 60 min.

Inhibitors-The mechanistic similarities in the hydrolytic pathways of sulfate and phosphate esters (24) prompted a quick test for any dual functionality in the sulfatase and phosphatase hydrolytic enzymes. At a concentration of lOWa M, p-nitrophenyl

Lfl

FIG. 2. Analysis of the sedimentation equilibrium ultracentrif- ugation of the purified arylsulfate sulfohydrolase II. C, con- centration of the enzyme in arbitrary units, at a distance X from the axis of rotation. The enzyme, 0.4 mg per ml, was centrifuged at 20’ and 14,000 rpm for 20 hours in 0.05 M NaOAc buffer, pH 4.8.

TABLE III Amino acid analysis

Amino acid Relative molar quantities” No. of residues per moleculP

Aspartic. ............. 3.48 Threonine .......... 2.044 Serine. ............... 2.08c Glutamic ............ 2.26 Proline .............. 1.20 Glycine .......... 2.54 Alanine 2.11 Half-cystine. ......... 0. 191c Valine .............. 1.59 Methionine ........... 0.530 Isoleucine ............ 1.23 Leucine .............. 1.77 Tyrosine. ........... 1.06~ Phenylalanine ........ 1.00 Ammonia. .......... 5.2Qc Lysine ............... 1.18 Histidine ............. 0.424 Arginine ........... 0.590 Tryptophan ........... Not determined

81 47 48 52 28 59 49 4

37 12 28 41 25 23

123 27 10 14

(1 Relative to phenylalanine. b An enzyme molecular weight of 65,000 was used in calculating

the residues per molecule. c Approximate corrections for decomposition of amino acids in

24 hours of hydrolysis were 5$$ for threonine, cysteine, and tyro- sine and 10% for serine. A 5% decrease was made for ammonia to correct for the ammonia liberated by decomposition of serine and threonine (21).

phosphate was completely inactive as a substrate for the enzyme. Addition of a metal ion such as Zn++ failed to produce any significant change. However, at the same concentration p-NPP resulted in 59oj, inhibition of the activity toward p-NPS (10W3 M),

In order to compare this enzyme with others which have been

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4930 Arylsulfatase from A. oryzae: Purijication and Properties

TABLE IV

Vol. 246, No. 16

Kinetics of arylsulfatase hydrolysis of substituted aromatic esters

At pH 4.02, p = 0.2, 0.2 M acetate buffer, 37.5”.

Ester V m*x

/Lmoles/min x 10”

4-Nitrophenyl. . . . . . . . . . . . . . . . . 1.59 f 0.070 3-Nitrophenyl. . . . . . . . . . . . . . . . . . 1.43 f 0.023 2-Nitrophenyl. . . . . . . . . . . . . . . . 1.70 f 0.010 2,4-Dinitrophenyl . . . . . . . . . . . . . 1.42 i 0.052 5-Nitrocatechol. . . . . . . . . . . . . . . 1.44 f 0.27 l-Chloro-4-nitrophenyl.. . . . . . . . 1.70 * 0.10 Salicyl . . . . . . . . . . . . . . . . . . . . . . . . . 1.62 & 0.15

0 pK, values taken from Reference 26.

KNI Vm,x/&n

M x 101 fimoles/min hf

41.9 f 7.8 3.80 f 0.87 15.3 f 3.3 9.35 f 2.15 9.07 f 0.04 18.8 f 0.20 3.17 i 0.04 44.9 f 2.2 37.7 f 5.5 8.83 i 1.2 4.59 f 1.0 37.0 32 1.05

142 i 33 1.14 f 0.37

P&Z’=

7.15 8.39 7.21 3.92

-7.7 5.33

12.38

TABLE V Kinetics of arylsulfatase hydrolysis of substituted phenyl sulfate esters

At pH 7.54, p = 0.2, 0.2 M Tris hydrochloride, 37.5”.

Substrate Vl?lSX K#l Vm.x/Km

pmoles/minX 108 M x 101 &e?&oles/min M

4-Nitrophenyl. . . . . . . . . . . . . . . . . 6.46 rt 0.59 18.18 & 2.56 0.355 f 0.082 2-Nitrophenyl. . . . . . . . . . . . . . . 5.98 f 0.14 4.22 zk 0.38 1.40 f 0.16 2,4-Dinitrophenyl. . . . . . . . . . . . . . 3.61 i 0.54 1.38 f 0.39 2.61 f 1.1 1-Chloro-4-nitrophenyl.. . . . . . . . . 4.90 & 0.27 3.65 f 0.47 1.34 f .25

pKa'=

7.15 7.21 3.92 5.33

a pK. values taken from Reference 26.

.5 E 2Hydroxy- \ ii 3.0- 5 Nitro

z 2.4 Dinitro I 4 Nitro 3

I ! =

Nitro 2 Carboxy E I I

2 Chloro - 2 Nitro

;: 4 Nitro i I

>E -I-

z

.,_25Chloro-4 Nitro

2.4 b,_ -I Dinitro I --It: Nitro

4 Nitro 2.0 I I I I I I I I I I

3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0

PKa

FIG. 3. Dependence of log V,,, on pK, of leaving group for substituted phenyl sulfates. Conditions were pH 4.02, 0.2 M

sodium acetate buffer, p = 0.2. Inset, plot of values obtained at pH 7.54,0.2 M Tris hydrochloride buffer, p = 0.2. The error flags represent the standard errors as determined from program HYPER.

reported to hydrolyze p-NPS, the inhibitory effects of phosphate, sulfate, sulfite, cyanide, fluoride, and chloride were examined (25). At concentrations of lO+ M, sulfate, chloride, and cyanide showed no inhibition while fluoride effected 69% inhibition and phosphate gave 44% inhibition. The inhibitory effects of fluoride and phosphate were considerably reduced on dilution and at higher substrate concentrations. Sulfite at 1OW M completely inhibited the enzyme activity.

The enzyme contained no essential metal which was extractable with 0.01 M EDTA on exhaustive dialysis for 19 hours. The EDTA-treated enzyme had a specific activity of 1.65 compared

with a value of 1.53 for an identical untreated sample. At a concentration of lop3 M, none of the metals added caused any activation of the enzyme above the level of the control. The monovalent cations (Na+, K+, NHd+) had no inhibitory effects; Zn++, Ca++, and Cu++ effected 30% inhibition while Pb++ and Al+++ inhibited the activity by 50%.

Kinetics-The hydrolysis of arylsulfate esters catalyzed by arylsulfatase obeyed the Michaelis-Menten rate law (Equation

1). The parameters derived from initial rates at pH 4.02 and pH 7.54 are given in Tables IV and V.

V,,XS v=S (1)

The reactions of all esters were studied at fixed enzyme concentra- tion and varying concentration of ester. No deviations were found graphically plotting the data as l/v against l/S.

Since at present there is no direct method for determining the active site concentration for arylsulfatase it was imperative that values of V,,, be measured with the same enzyme concentration. To maintain a fixed enzyme activity, velocities at a substrate concentration of 1.0 X 1O-3 M were determined on the same day and values of v were converted to Vmax from the known K, of the substrate. Constants calculated in this manner agree very well with those values of V,,, obtained from the program HYPER for stock solutions of different activities but corrected to the same activity by using p-nitrophenyl sulfate as a standard reference (see “Experimental Procedure”).

Figs. 3 and 4 illustrate the dependence of log V,,, and log V,,,/K,,, on the pK, of the corresponding phenol. The results show that log V,,,/K, decreases with increasing pK, of the substituent group while the plot of log V,,, against pK, is invariant relative to the pK, of the corresponding phenol.

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2.4 Dinitro

\ 2 Chloro- 4 Nitro

\ \

‘y Nitro

I I ,I-\ I I I I I I 3 4.0 5.0 6.0 7.0 8.0 9.0 10.0 II.0 12.0 13.0

PKo

FIG. 4. Dependence of log V,,,/K, on pIZ, of leaving group for substituted uhenvl sulfates. Conditions and symbols are the same as those described in legend t,o Fig. 3.

Since the values of Vm,, for the various substrates have been determined with one concentration of enzyme, V,,, serves as relative measure of k,,t.

DISCUSSION

Cherayil (5) demonstrated that A. oryzae grown on moist wheat bran produces three arylsulfate sulfohydrolases in propor- tions which vary with the cultural conditions. The isoenzymes, designated as I, II, and III on the basis of their electrophoretic mobilities, are separable by electrophoresis or ion exchange chromatography and are distinguished by different ratios of activity at pH 5.8 and 3.9. The fastest moving component, I, has an activity ratio of 7.0; Component II, of intermediate mobility, has an activity ratio of 1.1; and the slowest moving component, III, has an activity ratio of 2.7.

Growth on moist wheat bran in the presence of urea and diam- monium phosphate normally produces isoenzymes II and III but the proportion of isoenzyme II can be increased by growing the mold at lower levels of water. A partial purification and charac- terization of isoenzyme II have been described by Cherayil. In this study, the cultural conditions which produce predomi- nantly isoenzyme II have been exploited. Furthermore, it has been observed that mold bran which has partially dehydrated yielded a greater proportion of isoenzyme II over isoenzyme III.

The precipitation with acetone has been effective in concen- trating the enzyme from a large volume of the mold extract and, also, in preserving its activity until sufficient powder has been accumulated for use in the purification. The next step takes

advantage of the elution characteristics of isoenzymes II and III on ion exchange chromatography. On the Bio-Rex 70 column, isoenzyme II is eluted at a lower ionic strength than isoenzyme III, and a complete separation of the small amount of isoenzyme III is accomplished. Isoenzyme II is further purified by chromatography on Sephadex G-200 and preparative disc electrophoresis. The activity ratio of the purified enzyme is 1.1 and is consistent with that reported above for isoenzyme II.

After a 26-fold purification, isoenzyme II appears to be homo- geneous by the highly selective method of polyacrylamide disc gel electrophoresis. A high degree of homogeneity is also suggested by the single, symmetrical, active protein peak observed on sucrose density gradient centrifugation and the linear plot of In (? against X2 obtained from the sedimentation equilibrium run.

The amino acid composition of isoenzyme II bears a striking resemblance to that reported by Nichol and Roy (27) for aryl- sulfatase A from ox liver owing to the preponderance of glycine, aspartic acid, and glutamic acid. The relatively high content of these amino acids is reflected in a rather small partial specific volume of 0.722 and in a suspected low isoelectric point for isoenzyme II.3 A partial specific volume of 0.700 from the amino acid analysis and an isoelectric point of 3.6 have been reported for arylsulfatase A (27). Using a partial specific volume of 0.722, the apparent molecular weight of isoenzyme II is 65,000 f 2,000. Drnec (28) has reported an isoelectric pH of 4.23 and a molecular weight of 75,300 f. 7,000 for isoenzyme II.

The purified enzyme is inhibited by sulfite, fluoride, and phos- phate but not by sulfate, chloride, and cyanide. On the basis of the classification suggested by Dodgson and Spencer (25), it is neither type I nor type II so that this classification apparently applies only to mammalian systems. At pH 4.8 p-NIP acts as a competitive inhibitor of the enzyme activity toward p-NPS. It is interesting to note, however, that at pH 7.5 p-NPP is not an effective inhibitor of either the A. oryzae or Aerobacter aero- genes enzyme (29). This aspect is presently being pursued.

The effect of structure upon reactivity as determined by a plot of vmx against pK,, Fig. 3, reveals that the rates are completely insensitive to the pK, of the leaving group within the statistical error limits. Recent evidence with a partially purified prepara- tion of isoenzyme II (30) likewise has shown that in the acidic region of activity the substrates p-nitrophenyl- and 5-nitro- catechol sulfate were hydrolyzed at approximately equal rates. The effect of structure upon K, at pH 4.02 is indicated in Table IV and graphically illustrated in Fig. 4 where a plot of log V,,,/ Km against pK, is shown. These data suggest that the changing reactivity of the ester is reflected in K, rather than V,,,.

These results may be considered in terms of a multistep model developed to explain the kinetic behavior of certain classes of proteolytic and hydrolytic enzymes (31-33).

K. k2 k3 E$-SW ES - ES’ - E + Pz

+ (2) PI

where E is enzyme, X is substrate, ES is the adsorptive enzyme- substrate complex, ES’ is the product of the reaction of substrate with the enzyme releasing 1 mole of product, Pi, per mole of enzyme, and PZ is the second product resulting from hydrolytic

3 No migration of the enzyme was detected on gel electrophoresia at pH 4.5 under the conditions described by Williams and Reisfeld (16) and at different electrophoretic running times.

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4932 Arylsul$atase from A. oryzae: PuriJication and Properties Vol. 246, No. 16

2.4 Dinitro IyIChloro-4 Nitro

2 Hydroxy -5 Nitro

i 2 Carboxy

I I _ ^ ̂ YL

1.0 z.0 5.”

-Log KH + (min-‘)

FIG. 5. Log V,,JKm for the hydrolysis of aseries of substituted phenyl sulfates as a function of their rate of acid hydrolysis in 1 M HCI at 37.5”. Symbol conventions are those described in the legend to Fig. 3.

cleavage of the ester. According to this model Km and kcat = ((Vm,,) /E) are given by

k kzk3 cst = kz

and

K&a K,,, = -

x-2 + Ic3 (4)

If kB is rate-determining then Ir max is constant since a common intermediate will be hydrolyzed in each instance. However, irrespective of any relative magnitude placed on k3 and kz, the term Vm,,/K,,, = ka/K, will generally provide a useful kinetic constant measuring substrate effects on the binding constant or the rate step kz (34).

The results of this study indicate that the reaction of aryl- sulfate sulfohydrolase II with substituted phenyl ester sub- strates may fall into the class of enzyme kinetics given by Equa- tion 2. The existence of a linear (1: 1) correlation between log V,,,/K, (kcst E/Km) and log ku+ (Fig. 5) suggests that the effect of pK, is manifested in kz and not K,. Values for lcn+ were estimated from the structure-reactivity correlation of Benkovic and Dunikoski (4) extrapolated to 37.5”. A similar correlation has been established for the rates of the papain- catalyzed hydrolysis of a series of acyl esters and the correspond- ing rates for hydroxide-catalyzed hydrolysis (35). Deviations from the correlation of Fig. 5 include the esters of p-nitrophenyl-, 5-nitrocatechol, and salicyl sulfates.

The discrepancy with 5-nitrocatechol and salicyl sulfates may be attributed to the incursion of intramolecular group participa- tion in the nonenzymic rate (8) ; no rationale is presently available for the p-nitrophenyl sulfate behavior. The fact that a correla-

tion does exist in Fig. 5 mitigates against K, being dependent on P&. It is obvious that no similar relationship exists between log Fi,,, and log ku+. A similar correspondence between K, and kn+ for the arylsulfatase from A. metalcaligenes has pre- viously been noted (2).

These data do not uniquely exclude the possibility that the constant Vmax results from a rate-determining conformational change necessary for product deabsorption. Thus the existence of a sulfuryl enzyme intermediate although strongly suggested by the data is not rigorously required. If indeed a sulfuryl enzyme intermediate exists then lcz probably involves an acid-catalyzed transition state with considerable sulfur trioxide character (36).

Insets in Figs. 4 and 5 indicate that at pH 7.5 V,,, is now dependent on the pK, of the departing group. In terms of mechanism (2), sulfation or kl would now be partially rate- determining. It is obvious, however, that further speculation on the mechanism of action of this enzyme requires direct proof of the existence of the intermediate. Experiments of this nature are now in progress.

Acknowledgments-We wish to thank the Department of Biochemistry for the use of its research facilities in the enzyme purification, Dr. J. J. Ellis for the culture strain of A. oryzae, Mr. J. M. Schorr for the sedimentation equilibrium run, and Mrs. R. P. Sampson for the amino acid analysis. We also thank Dr. A. T. Phillips and Dr. H. R. Knull for their helpful advice.

REFERENCES

1. ROY, A, B., AND TRUDINGER, P. A., The biochemistry of in- organic compounds of sulfur, University Press, Cambridge, 1970, p. 133.

2. DODGSON, K. S., SPENCER, B., AND WILLIAMS, K., Biochem. J., 64, 216 ‘(1956).

3. JERFY, A., AND ROY, A. B., Biochim. Biophys. Acta, 175, 355 (1969).

4. BENKOVIC, S. J., AND DUNIKOSICI, L. K., JR., Biochemistry, 9, 1390 (1970).

5. CHER,IYIL, J. D., Ph.D. dissertation, St. Louis University, 1963.

6. BURICHARDT, G. N., ‘\ND WOOD, H., J. Chem. Sot., 141 (1929). 7. FENDLER, F. J., AND FENDLER, J. II., J. Org. Chem., 33, 3852

(1968). 8. BENKOVIC, S. J., J. Amer. Chem. Sot., 88, 5511 (1966). 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,

R. J., J. Biol. Chem., 193, 265 (1951). 10. HUGGINS. C.. AND SMITH. D. R., J. Biol. Chem.. 170, 391

(1947).’ ’ 11. CLELAND, W. W., Advan. Enzymol., 29, 1 (1969). 12. WILIUNSON, G. N., Biochem. J., 80, 324 (1961). 13. JOVIN. T., CHRAMBACH, A., AND NAUGIITON, M. A., Anal.

Biochem:, 9, 351 (1964). 14. Buchler instruction manual. Buchler Instruments, Inc., Fort

’ Lee, New Jersey. 15. ORNSTEIN, L., AND DAVIS, B. J., Ann. N. Y. Acad. Sci., 121,

321 (1964). 16. WILLIAMS, D. E., AND REISFELD, R. A., Ann. N. Y. Acad.

Sci., 121, 373 (1964). 17. CHRAMBACH. A.. REISFELD. R. A.. WYCKOFF, M.. AND ZAC-

CARI, J., Anal: Biochem.,20, 150’(1967). 18. MARTIN, R. G., AND AMES, B. N., J. Biol. Chem., 236, 1372

(1961). 19. PAZUR, J. H., KLEPPE, K., AND ANDERSON, J. S., Biochim.

Biophys. Acta, 65, 366 (1962). 20. YPHANTIS, D. A., Biochemistry, 3, 297 (1964). 21. MOORE, S., AND STEIN, W. H., in 8. P. COLOWICK AND N. 0.

KAPLAN (Editors), Methods in enzymology, Vol. VI, Aca- demic Press, New York, 1963, p. 819.

22. VAN KLEY, H., AXD BARTHOLOME, D., Fed. Proc., 28, 3414 (1969).

by guest on June 22, 2018http://w

ww

.jbc.org/D

ownloaded from

Issue of August 25, 1971 S. J. Benkovic, E. V. Vergara, and R. C. Hevey 4933

23.

24.

25.

2G.

27. 28. 29.

COHN, E. J., AND EDSALL, J. T., Proteins, amino acids, and peptides, Reinhold Publishing Corporation, New York 1943, p. 370.

BENKOVIC, S. J., AND BENKOVIC, P. A., J. Amer. Chem. Sot., 88, 5504 (1966).

DODGSON, K. S., AND SPENCER, B., Methods Biochem. Anal., 4, 234 (1957).

KORTUM, G., VOGEL, W., AND ANDRUSSOW, K., Dissociation constants of organic acids in aqueous solution, Butterworth, London, 1961.

NICHOL, L. W., AND ROY, A. B., Biochemistry, 4, 386 (1965). DRNEC. J. F.. Ph.D. dissertation, St. Louis University, 1968. FOWLED, L. ‘R., AND R~~MMLER,‘D. H., Biochemistry,’ 3, 230

(1964).

30. PRESSLITZ, J. E., Ph.D. dissertation, St. Louis University, 1968.

31. JF,NCKS, W. P., Catalysis in chemistry and enzymology, McGraw- Hill Book Company, Inc., New York, 1969.

32. GUTFR~UND, H., AND STURTEVANT, J. M., Proc. Nat. Acad. Sci. 77. S. A., 42, 719 (1956).

33. BENDER, M., KEZDY, F., AND WEDLER, F., J. Chem. Educ., 44, 84 (1967).

34. BENDER, M., AND KEZDY, F., Annu. Rev. Biochem., 34, 49 (1965).

35. KIRSCH, J. F., AND IGELSTR~M, M., Biochemistry, 6, 783 (1966).

36. BENKOVIC, S. J., AND HEVEY, R. C., J. Amer. Chem. Sot., 92, 4971 (1970).

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Stephen J. Benkovic, E. V. Vergara and R. C. HeveyAspergillus oryzaePurification and Properties of an Arylsulfatase from

1971, 246:4926-4933.J. Biol. Chem. 

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