Substrate Selectivity in the Action of Alkaline and Acid ...

Substrate and Acid

Selectivity in the Action Phosphatases *

of Alkaline

(Received for pltblication,

Prom

the Department

of Biochemistry

and Biophysics,

SUMMARY

The distinction between acid and alkaline phosphatases is based upon the marked difference in the 1)H ranges in which these enzymes are active (1, 2). Recent studies, however, have *This work was supported in part by Grant A-003 of the Robert, A. Welch Foundation, Houston, Texas. 1 On leave of absence from the Department of Biophysics, Weizmann Inst,itrlte, Rehovoth, Israel, where part of this work was done.

College Xtation,

19, 19(i8)

Texas 77843

suggested the existence of profound differences in their substrate specificities. Neumann, Uoross, and Katchalski (3) showed that alkaline phosphatase from Escherichin coli hydrolyzes X-substituted monoesters of phosphorothioic acid at rates similar to the hydrolysis of O-substituted monoesters of orthophosphoric acid. Recently, Eckstein (4) showed that O-substituted drrivativa of nucleotidcs of 1)hosphorothioic acid were hydrolyzed by acid phosphatase, but were not susceptible to hydrolysis by alka,line phosphatasc. Kinetic data were not presented in his studies. This paper describes the results of experiments designed to elucidate further the activity of these enzymes by testing activities of acid and alkaline phosphatases from several sources upon both 0- and S-substituted monoesters of phosphorothioic acid and O-substituted monoesters of orthophosphoric acid. The study presented here was prompt’ed by the observation that the S-substituted monoesters of phosphorothioic acid were completely resistant to enzymic hydrolysis by acid phosphata,ses. Furthermore, when the enzymic hydrolysis of the O-substituted monoesters of phosphorothioic acid was investigated, it became evident that they serve as inhibitors for alkaline phosphatase. On the other hand, the same types of esters (O-substituted monoesters of thiophosphoric acid) were hydrolyzed by acid phosphatases from several sources at a rate comparable to that shown with the 0-subst,ituted monoesters of orthophosphate. EXPERIMESTAL

PROCEDURE

Xaterials Purified enzyme preparations of alkaline phosphatases from E. coli and from bovine kidney, and acid phosphatases from wheat germ, potato, and bovine prostate gland were purchased from Worthington. p-Nitrophenyl phosphate was purchased from Sigma; 5,5’-dithiobis (2-nitrobenzoic acid) was purchased from Aldrich. Cysteamine S-phosphate, N-acetylcysteamine Xphosphate, trisodium phosphorothioate, barium X-(carboxymethyl) phosphorothioate, and disodium S-[2- (methoxycarboxyl)ethyl] phosphorothioate were prepared and purified according to the literature by methods reported in Reference 3. The O-substituted monoesters of phosphorothioic acid were synthesized from thiophosphoryl trichloride (5) and the corresponding alcoholate. The monoester was preferentially obtained

4671

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Alkaline phosphatase of intestinal origin hydrolyzed the S-substituted monoesters of phosphorothioic acid of the type RSP03Na2 (R = -CH2CH2NH2, -CH,CH,NHCOCH,, -CH,COO, or -CH2CHsCOOCzHs) at the S-P bond to yield orthophosphate and the corresponding thioalcohols. The rate of enzymic hydrolysis of cysteamine S-phosphate was measured at pH 9.0 in 0.1 N sodium barbital buffer at different concentrations of substrate and MgC12. The K, as well as the amount of MgC12 and V,,, values obtained, required for complete activation of the enzyme, were similar to the corresponding values obtained when p-nitrophenyl In marked contrast, phosphate was used as the substrate. however, O-substituted monoesters of phosphorothioic acid of the type ROPOeSKH (R = -CH3, -CH&H,, or -aNOe) were completely resistant to hydrolysis by alkaline phosphatases from Escherichia coli and from The O-substituted monoesters of thiophosphoric intestine. the enzymic hydrolysis of both acid (10M7 M) inhibited and p-nitrophenyl phosphate cysteamine S-phosphate (10e3 M) at a lo-fold concentration of the enzyme. Acid phosphatases from wheat germ, potato, and prostate gland did not hydrolyze S-substituted monoesters of phosphorothioic acid at detectable rates, but did hydrolyze O-substituted monoesters of phosphorothioic acid at rates comparable with those obtained when p-nitrophenyl phosphate served as the substrate for these enzymes. These findings suggest that acid and alkaline phosphatases act by two different mechanisms.

Texas A and AT University,

April

4672

Substrate

Selectivity

01 Alkaline

Phosphatases

Vol. 243, No. 18

Corrections were made for changes in E with pH and ionic strength. With cysteamine S-phosphate as the substrate, assays were performed by determining the concentration of the hydrolysis product, cysteamine. This det,crmination was done with 5,5’-dithiobis(2-nitrobcnzoic acid) according to the procedure of Ellman (11). dbsorbance measurements were made at 5-set intervals for 5 min, the values obtained wcrc plotted against time, and the velocities were derived from the linear part of the curves. No temperature control was provided, and all measuremcnts were carried out at roonl temprrature. Gel Filtration-Scl)hades G-100 in a column, 1.5 X 60 cm, was used to separate alkaline phosphatase (intestinal) labeled with 0-p-nitrophenyl thiophosphate. Elution was accomplished with 0.1 AI Tris-HCl, pH 8.5. The protein concentration of the solution of alkaline phosphatase was calculated from its absorption at 280 m/* with the use of a molar absorption, E, of 70 x 103. For the estimation of the amount of phosphorothioate derivatives bound to the protein, 0.1-d samples of the effluent wcrc plated on planchets, dried, and coulltcd for 1 to 5 min in a SuclcarChicago gas flow counter equipped with a11 automatic sample changer and a digital recorder. Concentrations of the radioactive material were calculated by comparison with known standard, prepared, plated, and counted in the same mamier and at the same time as the unknolvn solutions. RESULTS

(=eneruZ-l\Ieasurcmcllts of pH were ma& with a Radiometer model 22 l)H meter, equipped with a type G-202B glass electrode and a type K4312 c*alomcl electrode. Spectrophotometric measurcmcnts were made with a Zeiss model 1’11Q spectrophotometer, in which quartz cuvcttrs with a light path of 10 mm were used. Electrophoresis-High voltage electroyhoresis was performed on lJThatman xo. 1 paper at 36 volts per cm in an apparatus of the type described by Michl (7), with the use of pyridine-acetatewater buffer, pH 6.5, according to the method of Rgle et al. (8). For the detection of compounds possessing free amino groups, the elcctrophorctograms Jv-ere stained with ninhydrin (0.5cj,, w/v, in acetone containing 20 ml of w&r, 2 ml of concentrated pyridine, and 478 ml of acetone); for the dctcction of organic phosphate, inorganic phosphate, and phosphorothioic acid, the reagent of Fiske and SubbaRow (9) was used. Radioactive clcctrophoretograms containing Y’- or 3Wabelcd compounds were subjected to direct scanning with the use of an &1ctiograph II, SuclearChicago, model L-IOOA. The concentration of inorganic phosphate or thiophosphate was estimated by comparison with a known standard solution. Enxyrnic Activity-IXrcct qualitative evidence for the hydrolysis of the compounds tested with the various enzyme preparations was obtained with the use of the high voltage paper electrol)horcsia technique described previously (3). Kinetic stud& were performed spectrophotometrically by measuring Ihc amount, of each alcohol product, liberated with time. Inasmuch as p-nitrophenol was the l)roduct of hydrolysis of both p-nitrophenyl I)hosphutc and O~p~nitrophenyl thiophosphate, its liberation was estimated by reading the absorbance at 400 mp in the pH range, 7 to 9.5, where E = 17.2 X IO3 (3, lo), and at 330 rnp in the pH range, 4 to 7, where E = 6.0 x 103.*

Hydrolysis of Various 0- and S-Substituted Monoesters of Phosphorothioic Acid by dlkaline and Acid Phosphatases-Enzymic hydrolysis of the various substrates by acid and alkaline phosphatases was shown dircctlS by qualitative identification of the products after high voltage clcctrophoresis separation. To facilitate the identification of the hydrolysis products, 32Plabeled compounds were used for the S-substit,uted monoester derivatives, where one of the expected products of hydrolysis was inorganic orthophosphate. On the other hand, %labeled compounds were used with the 0-substituted monoesters of phosphorothioic acid. In the latter case, one of the products of enzymic hydrolysis-as was indicated in preliminary csperiments-was thiophosphate. The appropriate labeled com~OUJKIS were incubated with alkaline phosphatase in 0.10 M barbital buffer, pH 9.0, containing 1OP M MgCl,; substrate was present at a concentratioii of 1OF RI, and the concentration of alkaline phosphatasc was 10 pg per ml. Alfter the reaction mixtures had been incubated for 24 hours at room temperature, 25+1 aliquots xvere applied to Whatman Xo. 1 paper and subjected to high voltage elcctrophoresis for 30 min at pH 6.5, 35 volts per cm; the paper IT--as then scanned for radioactivity. When acid phosphatases from wheat germ, potato, and prostate gland were tested, similar procedures were used except that 0.1 M sodium acetate buffer, pH 5.5, was used and the enzyme concentration was 30 pg per ml. The results, summarized in Table I, show that all of the S-substituted monoesters of phosphorothioic acid were hydrolyzed by alkaline phosphntascs (E. coli and intestinal) to yield the corresponding thioalcohols and inorganic phosphate. Sone of the O-substituted monoesters tested was hydrolyzed to a detectable extent. 111 marked contrast, however, the acid phosl)hatases hydrolyzed all of the O-substituted monoesters of phosphorothioic acid to yield free alcohols and

1 The al~orhsncc of p-nitrophenol was measured in sodium acetate bluffer at various pI1 values: at 330 mp at constant ionic

strength (I’/2 = 0.1). The maximum ahsorption length yielded a molar absorption, E, of 6.0 X 103.

at this wave


by the use of a concentration of thiophosphoryl trichloride that n-as 2Oc/;, in excess of the alcoholate concentration. The compounds n-cre then isolated by column chromatography on J)owcx 50; the products so obtained were analyzed for their content of alcohol, phosphate, and sulfur. Finally, the monoester was crystallized as the monopotassium salt from a solvent system containing water and diosane. In the preparation of 32P- or Y?-labeled trisodium thiophosphate, cysteamine S-phosphate, and O-substituted monocsters of phosphorothioic acid, t,hiophosphoryl trichloride lab&d with either 321’ or 33S was used. ‘l’hc labeled thiophosphoryl trichloride compounds were prepared with the use of “?l’-labeled phosphorus trichloride or Wlabeled sulfur (5). Both labeled materials were obtained from the Radiochemical Ccntre, .1mer:-ham, England. The 3Wabeled compounds had specific activities ol 10 PC per pmole, whereas the 321-‘-labeled preparations had sl)ecific activities of 20 PC per pmole. Stock solutions of all substrates were prcl)arcd at a collccntration of 0.03 M in conductivity lvuter and stored in a freezer. h solution of 5,5’-dithiobis(2-nitrobenzoic acid) (2.0 mg per ml, in 0.05 JI sodium barbit,al buffer, pH 8.0) was kept at room temperature and was used within a felr- days after preparation; a solution of AIgC12 (0.3 RI) was also kept at room temperature. Sodium barbit,al-HCl and sodium acetate buffers wverc prepared according to the procedure of Dan-son et al. (6).

and Acid

Issue of September

H. Neumann

25, 1968 I

TABLE

Hydrolysis products of various 0- and S-substituted monoesters of phosphorothioic acid by alkaline and acid phosphatases The products were identified, after high voltage electrophoretic separation, by direct counting of the paper and locating the radioactive spot with the use of appropriate blank compounds. The electrophoretograms were also stained with phosphate reagent (9). p-Nitrophenol was located by its yellow color after spraying with dilute sodium hydroxide solution. Cysteamine was identified by ninhydrin reagent. Alkaline products

Substrate

phosphatase, identifieda

No No

cleavage cleavage

No

cleavage

‘, 32Pi

No

cleavage

No

cleavage

No

cleavage

0-p-Nitrophenyl phate

No

cleavage

3Y+Phosphorothioate,c a%-Phosphorothioate,~ 3%Phosphorothioate, p-nitro phenol Pi, p-nitrophenol

phosphate

p-Nitrophenol,

OD AT

ENZYMIC W*

ACTIVITY

moles/ml)

Pi

a Identical products were obtained with alkaline phosphatases from the various sources. b Identical products were obtained with acid phosphatases from the various sources. c The alcohol liberated was not identified.

thiophosphate. The acid phosphatases failed to hydrolyze any of the S-substituted monoesters of phosphorothioic acid, even to a small extent. Kinetics of Hydrolysis of Cysteamine S-Phosphate Catalyzed by in Barbital-Preliminary experiments Alkaline Phosphatase revealed that the rate of hydrolysis of cysteamine S-phosphate by alkaline phosphatase is dependent upon the concentration of MgC12 and upon the order in which the components of the mixture are added (Fig. 1). Experiments therefore were conducted to determine the concentration of h/lgC& above which no change in enzymic activity occurs. The enzyme concentration in these experiments was 3.0 pg per ml, the substrate concentration was of 5,5’-dithiobis(2-nitro2 x 10e3 M, and the concentration benzoic acid) was 4 X lop4 M. The concentration of MgClz was varied from 10m3 M to lo+ M. Measurements of absorbance at 412 rnp were made every 10 set during the first 6 min of the enzymic reaction. Identical experiments were done with the use of p-nitrophenyl phosphate (1O-3 M) as substrate under otherwise identical conditions. The results of these experiments are shown in Fig. 2, which indicates that a concentration of about 7 X 1O-3 M MgCl~ affords maximal activity. The effects of substrate concentration on the rate of enzymic hydrolysis of cysteamine S-phosphate by alkaline phosphatase were tested at pH 9.0 in sodium barbital buffer containing lo-* M MgC12. From the results of these experiments a Michaelis const,ant (K,) of 2.5 x 10V4 K’ and a maximum rate of hydrolysis (V,,,) of 0.7 x lo-* mole of substrate ml+ min-1 (pg of

2

I

3

TIME (IN MINUTES) 1. The dependence of enzymic activity on the order of The experiaddition of the components of the reaction mixture. lo’-% M ments were carried out at pH 9.0 in 0.1 M buffer containing MgCh, 10e3 M cysteamine S-phosphate, and 3.0 pg of enzyme per ml. Curve A represents the change in formation of p-nitrophenol with time when the reaction was initiated by the addition of the of p-nitrophenol as a substrat,e, Curve B shows the production function of time when the reaction was started by the addition of enzyme, and Curve C represents a control experiment in which no MgCIz was added. FIG.

6 Tc 5

”

12

3

4

5

6

7

8

9

IO

MOLAR CONCENTRATIONOF MgCl,x IO3 FIG. 2. The effect of the concentration of MgClp on the rate of hydrolysis of cysteamine S-phosphate (A) and p-nitrophenyl phosphate (B), catalyzed by kidney alkaline phosphatase at pH in 0.1 M 9.0. The reaction mixtures contained 0.002 M substrate sodium barbital buffer, pH 9.0, and 3 pg of enzyme per ml.


Cysteamine, 3”Pi N-Acetylcysteamine, “Pi c, 32Pi

p-Nitrophenyl

enzyme-l ml+) were calculated. Under identical conditions of pH, buffer, YIgClz concent.ration, and enzyme concentration no detectable activity was obtained when 0-p-nitrophenyl thiophosOn phate was tested for susceptibility to alkaline phosphatases.

Acid phosphatase products identified’

Cysteamine S-phosphate N-Acetylcysteamine Sphosphate S-(Carboxymethyl)phosphorothioate S-[2-(Methoxycarboxyl) ethyllphosphorothioate O-Methyl phosphorothioate O-Ethyl phosphorothioate thiophos-

4673

Substrate Selectivity of Alkaline and Acid Phosphatases

4674

OD AT

PMOLES

ENZYME

0016 0 015 0 014

OD AT 412mp

OD AT 28omu

c

t 4

20

15

1 IO

i

IO

20

30

40

NUMBER

50

60

70

80

05

i 90

OF TUBES

4. Gel filtration chromatographic pattern (on Sephadex G-100) of alkaline phosphatase. A 0.005.ml sample from each fraction was assayed at 22” for enzymic act,iviCy toward 2 X lo+ M cysteamine S-phosphate in 0.1 M sodilml barbital buffer, pH 9.0, containing 4 X IOP M 5,5’-dithiobis (2nitrobenzoic acid). The enzymic act,ivity was assayed 1)~ measluing the cysteamine formed by its reaction with the acid to prodlrce absorbance at 412 rnp. X--X, absorbance at 280 mp; l - - -0, enxymic act,ivity as reflected by absorbance at 412 mp. FIG.

0 013 0 012

I

0011 0010 0009 0.008

-1

2 & F

0007

"0 co

0006

z

0005

5

0004 0003 0002 0001 0

15

25

35

45

55

65

NUMBER OF TUBES,

75

85

95

Xl6ml

3. Chromatographic pattern on Sephadex G-100 of the reaction mixture containing alkaline phosphatase and O-p-nitrophenyl thiophosphate labeled with either Y? or 3%. From the absorbance of each fraction, measured at 280 m,~, the protein concentration was calculated (o- - -0). A O&ml sample from FIG.

each trtbe was plated material was calclrlated corrections for blauks;

and the concentration of the radioactive after counting and making appropriate radioactivity is shown as X--X,

280 ml* were tested for enzymic activity. Only the first peak contained enzymically active protein. Kinetics of Hydrolysis of 0-p-Sitrophenyl Thiophosphate by Acid Phosphatases-0-p-Nitrophenyl thiophosphatc ITas chosen for kinetic studies for two reasons: (a) one of its hydrolytic products, p-nitrophenol, could be measured spectrophotometrically with a high accuracy, directly in the reaction mixture, and (b) it is the closest, struct,ural analogue of the substrate usually used for measuring the activity of acid phosphatase (p-nitrophenyl phosphate). The reaction mixtures contained 2 X 10P3 M substrate (either 0-p-nitrophenyl thiophosphate or p-nitrophenyl phosphate), 10 Kg of acid phoxphatase (from potato or wheat acetate buffer, pH 5.6. The germ) per ml, and 0.1 M sodium reaction rates were determined by absorbance measurements at Rates of hydrol330 rnp t,aken at 30.set intervals up to 15 min. ysis were calculated from the linear portions of the curves, and were found to be essentially the same for the acid phosphatases from

both

potato

and

wheat

germ.

With

0-p-nitropheuyl

thiophosphate, 2) was 5.5 X 1OPO moles ml-l mirlP (pg of enzyme-’ ml-‘) ; under the same conditions the rate of hydrolysis of p-nitrophenyl phosphate was 7.2 X 10P” molts ml-l min+ (pg of enzyme-r nil-‘). Values for V,,, and K, were determined by varying the substrate concentrations over the range, 3 X 1OW M to 5 X IO+ M, velocit,ies were calculated from the linear parts of the time plots, and the data were plotted as l/v against l/s. The values for K, calculated for the two types of acid phosphatase were virtu-


the contrary, when small amounts of 0-p-nitrophcnyl thiophosphate (IOP to 1OP M) were added to the reaction mixture containing cysteamine S-phosphate (1OW M), alkaline phosphatase was found to be inhibited and no hydrolysis of cysteamine X-phosphate could be detected by high voltage electrophoretic analysis even after 24 hours of incubation at room temperature. Binding of 32P- or Wlabeled 0-p-Nitrophenyl Thiophosphate Phosph,alase-Alkaline phosphatase (30 mg, 0.2 to Alkaline pmolc) was dissolved in 3.0 ml of 0.1 M Tris buffer, pH 8.7, and was permit,ted to react with 0.4 ml of 1OP M 0-p-nitrophenyl thiophosphatc labeled with 3211 or with 3S. After incubation for 2 hours at room temperature, the reaction mixture, 3.4 ml in volume, n-as transferred to a Sephades (G-100) column and eluted with 0.1 M Tris, t)H 8.5, 1.5-ml fractions being collected. The amount of 0-p-nitrol)henyl thiophosphate bound to the protein was evaluated from optical density and radioactivit,y measurements, as described in “Experimental Procedure.” -1 typical gel filtration pattern is giveu in Fig. 3. The first peak at 280 mp had 1 eq of 9’ or 3% bound to the protein. No radioactivity was found in the other peaks, except some overlapping with the free 0-p-nitrophenyl thiophosphate. The separation of alkaline phosphatase by gel filtration under the conditious specified above was repeated in the absence of 0-p-nitrophenyl thiophosphate. Essentially the same pattern was obtained, as shown in Fig. 4. The three major peaks at

Vol. 243, n-o. IS

Issue of September

25, 1968

H. Neumann

4675


TABLE II ally identical; with 0-p-nitrophenyl thiophosphate as the substrate, the li, values were 2.2 x 1OP M and with p-nitroEnzymic hydrolysis of three types of subslrates by alkaline and acid phosphatase,s phenyl phosphate they were 2.5 x 1OP M. The values for V,, were found to be 3.2 x 1OPo moles ml-l mill-’ (pg enzyme ml-l) :1eavfor 0-p-nitrophenyl thiophosphate and 5.2 x 10-l moles 1~11~~ age >hown min+ (pg enzyme ml-‘) for p-nitrophenyl phosphate. Kinetic y high Enzyme and substrate oltsge experiments could not be performed for the enzymic hydrolysis xlper, lectroof cysteaminc S-phosphate by acid phosphatases because the horesi: (3) spontaneous hydrolysis of the substrate at pH 5.5 without added enzyme was identical with that produced by the addition of 30 M pg of arid phosphatase from potato, wheat germ, or prostat,e Alkaline phosphatase (E. gland per ml. The amouuts of cysteamine S-phosphate hycoli) drolyzed without enzyme and with enzyme were 2.0 x lo-l3 Yes 4.2 x 10-E Cysteamine S-phosphate.. 9.4 x 10-S moles per set and 2.2 x 1OP moles per set, respectively. p-Nitrophenyl phosphate. 5.8 x 10-X Yes 9.4 x IO-” p-Nitrophenyl thiophosphate. so Alkaline phosphatase (illThe data in Tables I and II reveal distinct differences in the testinal) substrate specificities of the alkaline and acid phosphatases, Yes 0.7 x 10-g 2.5 X 1OV Cysteamine S-phosphate.. although no differences were observed between similar enzymes Yes 1.1 x 10-S 2.5 x lO+ p-Xitrophenyl phosphate. from different. sources. As is shown in Table I, alkaline phosthio0-p-Kitrophenyl phatases hydrolyzed S-substituted monoesters of phosphorophosphate. No thioic acid and O-substituted monoesters of orthophosphoric Acid phosphatase (potato)* acid. The /cm values and the initial velocities of hydrolysis for No Cysteamine S-phosphate.. both substrates were essentially the same (Table 11). The p-Nitrophenyl phosphate. Yes 5.2 X IO-lo 2.5 X 1OP O-p-Sitrophenyl thiohydrolysis of the O-substituted rnonoestcrs of phosphorothioic phosphate . Yes 3.2 x 10-10 2.2 x 10-4 acid by alkaline phosphatases was not detected, and in fact one of thcsc coml)ounds (0-p-nitrophcnyl thiophosl)hatc) was obu Maximum rates of hydrolysis, V,,;,,, were expressed as moles The served IO br a potcnt inhibitor of alkaline phosphatases. of substrate hydrolyzed per ml per rnin per pg of enzyme per ml of inhibitioll of alkaline phosphatases was not att,ributable to the reaction mixture. The reactions were followed by spectrophotoremoval of nrctal ions (Zn ++ in the case of the enzyme from E. metric determination of the products. coli and 1\Ig’+ in the case of the intestinal enzyme) by O-pb Essentially identical results were obtained with acid phosnitrophenyl thiophosphatc inasmuch as other sulfhydryl comphatase preparations from wheat germ and from bovine prostate pounds, such as cysteaminc, were not inhibitory at an identical gland. concentration (1OP 1~). On the other hand, the acid phosphatases did not hydrolyze S-substituted monoesters of phosphoroity of the linked oxygen atom of the substrates for cnzymic thioic acid (‘Table I) but did hydrolyze the O-substituted monoactivities in which sulfur is incapable of serving. Another posacid under identical cxpcrimental esters of I)hosphorothioic sibility is that acid phosphatascs act at pH values where at least conditions. one of the hydrosyl groups of the phosphomonoesters is prescut The similarity of the hydrolysis of S-substituted monocsters in the undissociated form. If this is the case, the substitution of I)hosl,horothioic acid and of O-substituted monoesters of of a sulfur for an oxygen atom would be expected to influence orthophosphoric acid by alkaline phosphatases suggests that the ionization constant of the hydroxyl group of the substrate these enzymes do llot bind to the linking oxygen or sulfur atoms sufficiently to hinder the binding of the enzyme to the substrate, of these coml)ounds (3). Conversely, the lack of hydrolysis of and consequently to alter the enzymic activity. It is well the O-substituted monoestcrs of phosphorothioic acid by these known t,hat the substitution of a sulfur atom in acetic acid enzymes suggests that the two hydrosyl groups are essential for changes the pK, value from 4.6 to 2.2 (12)) and in phosphorothioic enzymic activity, and not even ont can be replaced by a sullhyacid all three pK, values are lower than the corresponding values dry1 group. HoTvcver, the possibility that only one hydroxyl in orthophosphoric acid (13). The pK, values of the fret hyfor the enzymic act,ivity and that the presgroup is iml)ortant droxyl groups are even lower when the sulfur atom of phosphoroence of a frc>e sulfhydryl in the O-substituted monocsters of thioic acid is present in an ester linkage.2 The hydrolysis of the phosphorothioic acid prevents their binding to the active site O-substituted monoester of phosphorothioic acid (O-p-nitroof the enzyme cannot bc excluded. The binding of O-p-nitrophenyl thiophosphate) by acid phosphatases may bc explained phenyl thiophosphate to alkaline 1)hosphatase was shown in the on the basis that the sulfhydq-1 group is ionized and thus retards experiment in which 0-p-nitrophcnyl thiophosphate, labeled the ionization of the free hydroxyl group, which in turn is availwith a2P or ssS, was incubated with the enzyme. After gel able to react with the enzyme. Furt,hermore, it seems that acid filtration on Sephadcx G-100 had removed the unbound O-pphosphatase does not have a linking site for sulfhydryl groups. nitrophenyl thiophosphrtte, t,he enzyme was found to contain In contrast to the behavior of many alkaline phosphomonoesper mole both 321’ and %, in addition to 1 mole of p-nitrophcnol terases, acid phosphatases are not inhibited by metal-complexing of enzyme. The failure of acid phosphatase to hydrolyze S-substituted 2 I. Z. Sternberg, S. Rogozinski, and II. Kel~mann, data to be monoesters of phosphorothioic acid may be due to the essentialpublished.

4676

Substrate

Selectivity of Alkaline

agents (2) such as cthylenediaminetetraacetate and cyanide. Cysteine or reduced glutathione (2) and molecular hydrogen are likelvise Tvithout influence on the activity of acid phosphatases. Acknowledgmelzt-I am grateful to Professor Ephraim Katchalski, Head, Department of Biophysics, Weizmann Institute of Science, and to Professor J. i\/l. Prescott, Department of Biochemistry and Biophysics, Texas A and M University, for making possible and encouraging my present study. REFERENCES 1. STADTMAN, T. C., in P. D. BOYER, BLCK (Editors), The enzymes, Vol. Sew York, 19G1, p. 55. 2. SCHMIDT, G., in P. I>. BOYER, H. The enzymes, Vol. V, (Editors), Sew York, 1961, p. 37.

and Acid Phosphatases

Vol. 243, No. 18

II., BOROSS, L., AND KATCHALSKI, E., J. Biol. Chem., 242, 3142 (19GT). 4. ECKSTEIN, F., J. ilnzey. Chenz. Sot., 88, 4292 (19%). 5. KNOTZ. F.. ijsterreichische Chenz. Zeituna. 50, 128 (1949). G. D.i\vs&, &. M. c., ELLIOTT, D. C., E:LL;oTT, iv. il., AND JONES, K. M. (Editors), Data for biochemical research, Oxford University Press, London, 1959, p. 203. 3. NEUMANN,

7.

H., Monatsh. 82, 489 (1951). 8. RYLE, A. P., SANGER, J.. 60, 541 (1955). MICHL,

Chem.

Verwundte

F., SWTH,

Tiele

L. F. AED KITAI,

Anderer

Wk.,

R., Biochem.

9. FISKE, C. H.‘ A&

II. LARDY, AND K. MYRV, Ed. 2, Academic Press, asI> K. MYRBBCK Ed. 2, Academic Press,

TARDY,

Chem., 66, 375 Sunn,~Row, Y., J. Sol. (19251. Chem. Sot., 82, 1778 (1960). 10. JANCKS, W. A., J. Amer. 11. ELLMAN, G. L., Arch. Biochem. Biophys., 82,70 (1959). 12. REID, E. E., Organic chemistry of bivalent sulfur, Chemical

Publishing Company, New York, 1958, p. 436. 13. NEUMANN, EI., STEINRERG, I. %., AND KATCHALSKI, Amer. Chem. Sot., 87, 384 (1965).

E., J.


Substrate Selectivity in the Action of Alkaline and Acid Phosphatases Hava Neumann J. Biol. Chem. 1968, 243:4671-4676.

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