THE JOURNAL OF UIOLOGICAL CHE~CIISTRY Vol. 243, No. 13, Issue of July 10, pp. 3693-3701, 1968

Printed in U.S.A.

Inhibition of “Serine” Esterases by Phenylarsonic Acids

a-CHYMOTRYPSIN AND THE SUBTILISINS*

(Received for publication, April 3, 1968)

A. N. GLAZER

From the Department of Biological Chemistry, University of California Xchool of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, California 90024

SUMMARY

Chymotrypsin, trypsin, and Novo and Carlsberg subtilisins undergo a reversible, time-dependent inhibition by phenyl- arsonates. The inhibition of these enzymes by p-nitro-, p-tolyl-, p-aminophenyl-, and phenylarsonate was studied in detail, as a function of pH and inhibitor concentration, by a variety of approaches. KI values were determined for the inhibition of chymotrypsin and the subtilisins by these compounds. Active site titrations and dye displacement experiments were used to show that the inhibition results from the interaction of the arsenicals with the active sites of these enzymes. The pH dependence of the inhibition of both chymotrypsin and the subtilisins followed the theoretical curve for the protonation of a group on the enzymes with a pK’ value of 7.15. The results obtained in this study, taken in conjunction with earlier scattered reports in the literature, suggest that the phenylarsonates are general inhibitors of “serine” esterases. Possible mechanisms for such inhibi- tion are discussed.

The point of departure for the work presented here was the finding that 4-(4’-aminophenylazo)phenylarsonic acid interacted specifically and reversibly with Novo and Carlsberg subtilisins to yield an inactive 1: 1 enzyme-dye complex (I). This protein- dye interaction exhibited an unusually long time dependence for both the association and the dissociation processes. The dye neither interacted with nor inhibited trypsin or chymotrypsin. A study of compounds structurally related in various ways to 4-(4’.aminophenylazo)phenylarsonic acid showed that a variety of phenylarsonic acids inhibited the subtilisins and appeared to act by binding to the same site as the dye (1). These compounds also exhibited a time dependence for both inhibition and reactiva- tion. In contrast to the dye, however, smaller arsenic acids, such as phenylarsonic acid, were found to be highly potent reversible

* This investigation has been aided by Grant GM 11061 from the National Institutes of Health, United States Public Health Service.

inhibitors of chymotrypsin and trypsin as well as of the sub- tilisins.

A number of instances of inhibition of enzymes by pentavalent organic arsonates have been reported (2). These reports have not hitherto included chymotrypsin, trypsin, or the subtilisins. There is little clear cut information, however, as to the basis of such inhibitory effects (2). The trivalent arsenicals have been shown to react with either thiols or dithiols, i.e. their effec- tiveness as inhibitors appears to depend on reactions of the type

S-R’ /

R-As=0 + 2 HS-R’ ti R--As

\ S-R’

It has been suggested that the action of pentavalent arsenicals may be explained, in some cases, by a partial reduction to the trivalent form by any thiol groups present

R-As0 (OH) 2 + 2 HS-R’ = R-As==0 + R’-S-S-R’ + 2HzO

and that the effect of the arsenic acid is due to the arsenoxide produced in this manner (2,3). Clearly, this explanation cannot hold in the case of the inhibition of cr-chymotrypsin or the sub- tilisins by phenylarsonic acids. The first enzyme contains no thiol groups, and the subtilisins contain neither cystine nor cysteine (4).

In view of the paucity of information relating to the mech- anism of action of pentavalent arsenicals, in spite of their ex- tensive use as drugs and in research, an examination was under- taken of the interaction of these compounds with certain “serine” esterases. This paper presents the results obtained with CZ- chymotrypsin and the subtilisins.

EXPERIMENTAL PROCEDURE

Materials and Melhods-Lyophilized crystalline Carlsberg subtilisin, Batch 50624, and crystalline bacterial proteinase Novo, Batch 60, were obtained from Novo Industries, Inc., Copenhagen, Denmark. Stock protein solutions were prepared by dialysis against 0.2 M acetate-O.02 M cac12 at pH 6.0 at 4” (5). Protein concentrations in these solutions were determined spectrophotometrically with the use of an E:Fm (278 rnp) value

3693

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3694 Inhibition of “Serine” Esterases by Phenylarsonates Vol. 243, No. 13

of 11.7 for Novo subtilisin (6), and an E:zm (280 rnp) value of 8.6 for Carlsberg subtilisin (7). For experiments at other pH values, the enzymes were dissolved directly in the appropriate solution and the concentration of active enzyme was determined by spectrophotometric titration with N-truns-cinnamoylimidaa- ole (8). Three times crystallized, salt-free a-chymotrypsin, Lot CDI-7JC, was obtained from Worthington, and Lot 65639 from Calbiochem. These preparations contained 96.0 and 96.5 % active enzyme, respectively, as determined by titration with N-trans-cinnamoylimidazole (8). or-Chymotrypsin concentra- tion was determined spectrophotometrically with the use of an Et?,,, (280 mp) value of 20.0 (9). Crystalline, salt-free trypsin, Lot TRL BJB, was obtained from Worthington. Stock trypsin and cy-chymotrypsin solutions were prepared in 0.001 N HCl.

High purity phenylarsonic acid and p-tolylarsonic acid were obtained from Alfa. p-Nitrophenylarsonic acid and 4-(4’- aminophenylazo)phenylarsonic acid were obtained from Ald- rich; p-arsanilic acid was obtained from Eastman and from K

20 I I I 1 I

18

:

3

"0 IO 20 30 40 50

TIME (MINUTES)

FIG. 1. Kinetics of hydrolysis of N-acetyl-L-tyrosine ethyl ester (0.01 M) by a-chymotrypsin (7.1 X 1e4 mg per ml) in 0.1 M

CaC12 at pH 6.0 and 25” in presence and in absence of various phenylarsonates. Inhibitor concentration was 4 X 10-s M in all cases. - - -, extensions of the apparent zero order rates of sub- strate hvdrolvsis reached in the presence of the inhibitors. The inset shows the Lineweaver-Burk plot for the hydrolysis of N- a.cet,vl-L-tvrosine ethvl ester in 0.1 M CaClg at DH 6.0 and 37”: A, no inhibitor; B, in the presence of 2 X 10-3 & p-nitrophenyl- arsonate. V is the rate of substrate hydrolysis in milliequivalents per min per 5-ml assay mixture at an enzyme concentration of 4 X lo+ mg per ml; S is in moles per liter.

and K Laboratories, Brooklyn. N-trans-Cinnamoylimidazole, proflavine sulfate, and thionine were obtained from Mann, and Biebrich Scarlet from K and K. Recrystallized p-nitrophenol and p-nitrophenylacetate were obtained from Sigma.

Determination of E&erase Activity-The rates of hydrolysis of N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-arginine ethyl ester by the subtilisins, trypsin, and chymotrypsin were deter- mined with a Radiometer model TTTlc pH-stat equipped with a type PHA630T scale expander and a thermostated reaction vessel. The volumes of the reaction mixtures were 5 ml and titrations were performed at either 25” or 37” with 0.02 N NaOH as titrating agent. No buffers were used and assays were per- formed in 0.1 M CaClz unless otherwise specified.

Spectrophotometric Measurements-Measurements at a single wave length were performed with a Zeiss PM& II spectrophotom- eter. All other spectroscopic data were obtained with a Cary model 14 recording spectrophotometer.

RESULTS

E$ect of Phenylarsonic Acids on c&hymotrypsin-Examination of the initial rate of hydrolysis of ester substrates by cr-chymo- trypsin in the presence of phenylarsonic acids indicated these compounds to be potent inhibitors of the enzyme. Depending on the particular arsenic acid used, either the inhibitory effect could be seen immediately on addition of the enzyme, as with p-nitrophenylarsonate, or a rapid fall in enzymatic activity was observed, as with phenylarsonate, or there was a slow decrease in activity with time, the rate and extent of the decrease de- pending on temperature, substrate, and inhibitor concentration, as observed with p-tolylarsonate and p-arsanilate (Fig. 1). In all cases, an apparent zero order rate of substrate hydrolysis was attained when the assays had been allowed to proceed for a sufficient period of time, as shown in Fig. 1. It could readily be shown that the inhibition by phenylarsonates was of the strictly competitive type kinetically (Fig. 1).

Kinetics of Dissociation of cu-Chymotrypsin-Phenylarsonate Complexes-The slow attainment of equilibrium in the inhibition of c-u-chymotrypsin by certain of the phenylarsonates suggested that reversal of the inhibition might also be slow. This was examined by incubating oc-chymotrypsin (7 X 1OV M) with p- arsanilate (2 X lop3 M), p-tolylarsonate (2 X 10e3 M), p-nitro- phenylarsonate (2 X 1O-2 M), and phenylarsonate (2 X lop2 M)

in 0.1 M Tris-chloride-O.01 M CaClz at pH 7.0 and 25”. After 80 min, the chymotryptic activity of the incubation mixtures was assayed by dilution of a lo+1 aliquot of the incubation mixture into 5 ml of 0.01 M N-acetyl-L-tyrosine ethyl ester in 0.1 M CaCb

at pH 7.0 and 25” in the pH-stat. The time taken for the com- plete recovery of esteratic activity could be determined in this manner. Under these conditions, recovery of activity was instantaneous after inhibition with p-nitrophenylarsonate, and required 4, 6, and more than 80 min in the case of the phenyl-, p-tolyl-, and p-aminophenylarsonates, respectively. This experi- ment was repeated by assaying at both pH 6 and pH 8. The rate of reactivation was found to increase sharply with increase in pH. Thus, for p-arsanilate, the time taken for the recovery of full activity at pH 8.0 was less than 8 min, as compared with over 120 min at pH 6.0.

Indeed, the dissociation of the Lu-chymotrypsin-p-arsanilate complex at pH 6 and 25” was sufficiently slow to permit esti- mation of the amount of free enzyme present in the incubation mixture at any time by assaying at pH 6 under the conditions

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- I I I

40 80 120

TIME (MINUTES)

16C 1

FIG. 2. Kinetics of inhibition of a-chymotrypsin and trypsin by p-arsanilate. Incubation mixtures (a to d) contained chymotrypsin (0.48 mg per ml) in 0.1 M acetate-O.01 M CaClz buffer at pH 6.0 and 25”, and p-arsanilate at the following concentrations: a, 4.15 X 1OP M; b, 2.05 X lo+ M; c, 1.04 X 1OP M; and d, 2.05 X IO+ M +

1.25 X 1OP M hydrocinnamate. Mixture e contained trypsin (1.9 mg per ml) in the above pH 6.0 buffer at 25” and 5 X 1OP M

p-arsanilate. The extent of inhibition was determined as a function of time by removing 25-~1 aliquots of the incubation mixtures at suitable time intervals and assaying the init.ial rates of esterolysis of N-acetyl-L-tyrosine et,hyl ester (0.01 M) in the case of chymotrypsin and N-benzoyl-L-arginine ethyl ester (0.025 M)

with trypsin. The assays were performed in the pH-stat in 0.1 M CaClz in a total volume of 5 ml at pH 6.0 and 25”.

described above and determining the initial rate of esterolysis. The kinetics of inactivation of cl-chymotrypsin by p-arsanilate, as a function of inhibitor and enzyme concentration, was deter- mined in this manner, and representative results are shown in Fig. 2. Over an enzyme concentration range of 8 to 24 x 10-b M and a p-arsanilate concentration range of 1 to 5 X 10P4 M,

the initial rates of oc-chymotrypsin inactivation followed strictly second order kinetics, i.e. the behavior was consistent with the equation

E+I k 2nd - EI

No ‘Lsaturation” effect was observed over the inhibitor concen- tration range given above; higher inhibitor concentrations could not be used because the inactivation was too fast to follow ac- curately. However, addition of competitive inhibitors of 01- chymotrypsin, such as hydrocinnamate, produced a dramatic decrease in the rate of inactivation (see Fig. 2). This observa- tion, coupled with the competitive inhibition kinetics observed with the phenylarsonates, suggests a requirement for binding and indicates that the equation

fast slow E+I z===.= EI z=zzrd EIf

may be a more accurate representation of the inhibition process. In$uence of Phenylarsmates on Binding of Dyes to a-Chymo-

trypsin-Studies on the binding of proflavine (10-13) and Bie- brich Scarlet (14) to ar-chymotrypsin have shown that these dyes bind specifically and stoichiometrically to the active site

region. A binding site exists elsewhere on the molecule for the phenothiazine dye, thionine (15). The phenylarsonates were found to compete for the binding sites of both proflavine and Biebrich Scarlet, but had no influence on the binding of thionine (Figs. 3 and 4). The experimental conditions are given in the legends to Figs. 3 and 4.

The difference spectra obtained with mixtures of cy-chymo- trypsin and phenylarsonates with proflavine and Biebrich Scarlet exhibited perfect isosbestic points, i.e. only free dye and protein-dye complex were present; no evidence of ternary com- plexes of protein-phenylarsonate-dye was observed (Figs. 3 and 4). The above results are consistent with the protection by hydrocinnamate and substrate described above and give strong support to the conclusion that the phenylarsonates exert their inhibitory effect through direct binding at the active site of CZ- chymotrypsin. From the observation that thionine binding is essentially unaffected by these inhibitors, it is unlikely that the binding of phenylarsonates produces a profound conformational change.

The competitive displacement of proflavine from a-chymo- trypsin by the phenylarsonates provided a means for direct measurement of the Kr values for these compounds. The results are given in Table I.

Determination of K1 Values by Titration with p-lliitrophenyl- acetate-Advantage was taken of the very slow dissociation of the cr-chymotrypsin complexes with the phenylarsonates to measure the concentration of free enzyme present, at equilibrium, in the enzyme-inhibitor mixtures by titration with p-nitrophenyl- acetate. At pH 7, under the conditions used (see Fig. 5), the “burst” of p-nitrophenol representing the acylation reaction (16, 17) was over in less than 15 set and the subsequent rate of

+0.1

AA

-0. I

-0.2 380 400 420 440 460 480 500 400 420440 460 480 500

WAVELENGTH (mp)

FIG. 3. Displacement of proflavine from cu-chymotrypsin by phenylarsonates. A, difference spectra obtained by comparing the absorbance of a mixture of proflavine (1.75 X 1Om6 M) and 01- chymotrypsin (1.3 X lo+ M) with that of the dye alone, in the presence of Curve 1, no inhibitor; Curve 2, p-nitrophenylarsonate; Curve 3, p-tolylarsonate; and Curve 4, p-arsanilate, at an inhibitor concentration of 1.8 X 1OP M in each case. B, difference spectra obtained by comparing the absorption of a mixture of proflavine (1.75 X lo+ M) and cu-chymotrypsin (1.35 X 10e4 M) with that of the dye alone in the presence of Curve 1, no inhibitor; Curves 2, 3, and 4, phenylarsonate, 5 X 10e3 M, 1 X 10-Z M, and 2 X 10-z M, respectively. All spectra were obtained after incubation of the mixtures for 90 min at 25” in 0.073 M phosphate buffer at pH 7.0. In each case the reference dye solution contained an amount of the appropriate inhibitor matching that in the incubation mix- ture. The light path was 1 cm.

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3696 Inhibition of “Serine” Esterases by Phenglarsonates Vol. 243, No. 13

+0.3

+0.2

+O.I

540 560 580 600 620 640 660 680

WAVELENGTH (mpL)

FIG. 4. Effect of p-arsanilic acid on the binding of Biebrich Scarlet and thionine by oc-chymotrypsin. A, difference spectra obt,ained by comparing the absorbance of a mixture of Biebrich Scarlet (3.65 X 10m5 M) and ol-chymotrypsin (1.37 X IOW M) with that of the dye alone, in the presence of Curve 1, no inhibitor; Curve 2, 1.96 X 10-Z M p-arsanilate; and Curve S, 3.92 X 1W M p- arsanilate. All spectra were obtained after incubation of the mixtures in 0.078 M phosphate buffer at pH 7.0 and 25” until no further change in absorbance at 550 rnp was observed. B, differ- ence spectra obtained by comparing the absorbance of a mixture of thionine (1.8 X 10m5 M) anda-chymotrypsin (1.32 X 1OW M) with that of the dye alone, Curve 1, in the absence of inhibitor; Curve 2, in the presence of 1.82 X 10-s M p-arsanilate. The spectra were obtained after incubation of the mixtures for 210 min at 25” in 0.072 M Tris-0.007 M CaClz at pH 7.0. In each case, the reference dye solution contained an amount of p-arsanilate matching that in the incubation mixture. The light path was 1 cm.

p-nitropheaol formation was linear for at least 24 min in mixtures containing phenyl-, p-tolyl-, or p-aminophenylarsonate, as well as in the control solution (Fig. 5). Thus, an accurate extrap- olation could be made to zero time and the burst used to cal- culate the amount of free enzyme present and the K I. The experimental conditions and representative results are shown in Fig. 5. The K1 values (Table I) obtained in this manner were in good agreement with those calculated from proflavine

displacement. The dissociation of p-nitrophenylarsonate was too rapid to permit an evaluation of the KI by the above tech- nique. In the presence of 3.3 x 10P3 M p-nitrophenylarsonate at pH 7, acylation of the enzyme was complete wit#hin 15 sec.

pH Dependence of Inhibition of ol-Chymotrypsin by Phenyl- arsonatesThe inhibition of chymotrypsin was studied as a function of pH with N-acetyl-Myroeine ethyl ester as substrate. The same concentrations of substrate, inhibitor, and enzyme were maintained for assays over the pH range, 6 to 8.5. Since the

TABLE I

Dissociation constants of complexes of a-chymotrypsin with various phenylarsonates

All measurements were performed at pH 7.0 and 25”. For de- tails of experimental conditions, see the legends to Figs. 3 and 5.

I Kr, determined by

Inhibitor

Phenylarsonate ........... p-Nitrophenylarsonate. .... p-Tolylarsonate. .......... p-Arsanilate ..............

Titration with Displacement of P-nitrophenylacetatea proaavineb

‘W ‘W

6.88 x 10-z 6.5 X 1OW 1.04 x 10-S

7.98 x 10-d 6.41 X 1OW 2.76 X 1OP 2.02 x IO-4

Q In 0.092 M phosphate buffer. ) The calculations were based on cM at 444 mp for proflavine of

41,000, a Kdiss value for the a-chymotrypsin-proflavine complex of 2.5 X 1O-5 M, and ACM for the complex of 22,000 at 465 rnp deter- mined in 0.072 M phosphate buffer at pH 7 and 25”.

1.4 I I I I I

A I cm 410

5 l

0 I - T - 7 I

0 I 2 3 4 5

TIME (MINUTES)

FIG. 5. Reaction of a-chymotrypsin with p-nitrophenylacetate in the presence and in the absence of phenylarsonic acids. 01. Chymotrypsin (5.6 X 10m5~) in2.7 ml of 0.092 M phosphate buffer at pH 7.0 was incubated for 210 min at 25” in the presence of 1, no inhibitor; 2, 6.67 X lO-$ M phenylarsonate; S, 1 X 10-s M p-tolyl- arsonate; 4, 6.67 X lo-* M p-arsanilate. At the end of this time, 300~1 of 10 mM p-nitrophenylacetate in acetonitrile were added and the kinetics of appearance of p-nitrophenol was followed as shown above. Curve 5 represents the spontaneous rate of hydrolysis of p-nitrophenylacetate under the above conditions in the absence of enzyme or inhibitors. The phenylarsonic acids. at. the eoneen- trations given above, did not affect this rate.

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PH

1 I , ,Y- 6.0 6.5 7.0 7.5 8.0 8.5

PH

3 9.c

FIG. 6. pH dependence of the inhibition of a-chymotrypsin by p-nitrophenylarsonate. The degree of inhibition was determined by assaying the esteratic activit.y toward N-acetyl-L-tyrosine ethyl ester (0.005 M) in 0.1 M CaClz at 25”, as a function of pH, at an enzyme concentration of 2.5 X lo+ mg per ml, in the presence and in the absence of 4 X 1W3 M p-nitrophenylarsonate. The inset shows the pH dependence of the inhibition of or-chymotrypsin (5 X 1OP mg per ml) by phenylarsonate (2 X 1V M). All other conditions were as above, except that certain assays (0) were performed in 0.1 M KCI. Sufficient time was allowed in zssays in the presence of phenylarsonate for the attainment of apparent zero order rates of substrate hydrolysis (see text). The curves drawn are theoretical, as calculated for the protonation of a single group of pK’, 7.15.

I<, for N-acetyl-L-tyrosine ethyl ester, as for other aromatic acetylamino acid esters, shows only a small increase with pH over this range (18, 19), the observed percentages of inhibition serve as a measure of the variation of KI with pH. The data obtained are shown in Fig. 6.

It is of considerable interest that, with both inhibitors, the data obtained followed closely a theoretical curve for the pro- tonation of a group with a pK’, of 7.15. The PK’,~ of phenyl-

arsenic acid is 8.48 (20), and that of p-nitrophenylarsonic acid, 7.80 (20). Since the pH dependence of the inhibition is the same for the two compounds, it is clear that it does not result from the ionization of the inhibitor but rather from that of a group on the

enzyme. It may be added that Kr values were obtained for p-nitrophenylarsonate from competitive inhibition data over the pH range, 6 to 7.5, and followed the same curve as that fitted to the percentage inhibition values. The significance of the pH dependence is discussed more fully below in conjunction with the data on the subtilisins.

E$ect of Guanidine on Formation and Stability of cr-Chymo- trypsin-p-Arsanilate Complex-The details of the experimental procedure are given in the legend to Fig. 7.

It has been established by other investigators (21, 22) that exposure of or-chymotrypsin to concentrated solutions of guani-

dine at pH values near neutrality produces unfolding of the

molecule and loss of enzymatic activity. On adequate dilution of the denaturation mixture, refolding of the protein and full recovery of enzymatic activity take place (21, 22). This ob- servation was readily reproduced in the present, study (see Fig. 7, Curve C). When ol-chymot.rypsin was incubated at pH 6.0 with 0.005 M p-arsanilate and the activity was assayed in the pH-stat as a function of time, the initial rates of substrate hy- drolysis indicated 96.5% inhibition at 5 min and at 160 min (Fig. 7, Curve A). When, however, a mixture of cu-chymotrypsin and p-arsanilate was incubated for 5 min at pH 6 and then guani- dine hydrochloride solution was added to a final concentration of guanidine of 6.4 M, slow recovery of enzymatic activity was observed. Essentially full enzymatic activity was recovered upon dilution into the assay mixture after approximately 220 min of incubation in the presence of guanidine (Fig. 7, Curve B).

Thus, prolonged exposure to guanidine at neutral pH does result in dissociation of the cu-chymotrypsin-p-arsanilate complex. Addition of p-arsanilate to a solution of a-chymotrypsin in 6.4 M guanidine hydrochloride at pH 6 does not affect the recovery of activity upon adequate dilution, i.e. the results obtained are

IOC

80

60

40

20

0

I I I I I I I I I ,- A-----,-

I I I I I I I Ol 1 I

0 40 80 120 160 200

TIME (in minutes) FIG. 7. Effect of guanidine on the rate of reactivation of LY-

chymotrypsin inhibited by preliminary incubation withp-arsanilic acid. The following mixtures were prepared from a fresh stock solution of a-chymotrypsin (2.75 mg per ml in 0.001 N HCI) and a 0.2 M acetate-O.02 M CaClz buffer at pH 6.0. A: a 0.25-ml aliquot of cu-chymotrypsin solution was mixed with 0.25 ml of the pH 6.0 buffer containing 0.05 M p-arsanilic acid (adjusted to pH 6 with NaOH). After 5 min of incubation at 25”, 2.0 ml of the pH 6.0 buffer were added and the incubation was continued. Aliquots of the incubation mixture (25 pl) were assayed, as a function of time, in the pH-stat. The assay mixture consisted of 5 ml of 0.01 M N-acetyl-L-tyrosine ethyl ester in 0.1 M CaClz at pH 6.0 and 25". B: to a mixture of a-chymotrypsin and p-arsanilic acid prepared and incubated for 5 min exactly as in A above, 2.0 ml of 8.0 M guanidine hydrochloride in the pH 6.0 buffer at 25’ were added, and the activity was assayed as a function of time as described for A. C: a 0.25-ml aliquot of a-chymotrypsin solution was mixed with 0.25 ml of the pH 6.0 buffer. After 5 min, 2.0 ml of 8.0 M guanidine hydrochloride in the pH 6.0 buffer at 25” were added and the incubation was continued. The activity was assayed as a function of time as described above.

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3698 Inhibition of “Serine” E&erases by Phenylarsonates Vol. 243, No. 13

identical with those shown in Fig. 7, Curve C. Thus, the native conformation of the enzyme is required for inhibition by p- arsanilate.

The above results indicate that the cr-chymotrypsin-p-arsanil- ate complex is much more stable in guanidine than the active enzyme. Since acyl-chymotrypsins, with a variety of acyl groups on the serine residue, have been shown to possess a very much higher stability to denaturing agents than chymotrypsin itself (see Reference 23 for a review), this stabilization may be a property conferred by many compounds which interact strongly with the enzyme in the region of the active site.

Effect of Hydroxylamine-The presence of 0.5 M hydroxylamine in incubation mixtures containing p-arsanilate (0.002 M) and cr-chymotrypsin (3 X 1O-6 M) at pH 6.0 or 7.0 did not influence the extent of inhibition of the enzyme. Also, the presence of 0.05 M hydroxylamine in assay mixtures at pH 6 and 7 produced only a very slight (<lo%) increase in the rate of reactivation on dilution of the incubation mixture to a final p-arsanilate concentration of lop4 M.

Effect of Phenylarsonic Acids on Trypsin-The phenylarsonic acids inhibitory to chymotrypsin were found to inhibit trypsin as well. However, the rates of interaction of the various in- hibitors and their affinities for trypsin were different from those observed with chymotrypsin (see Fig. 2, Mizzture e, for example). The interaction of the phenylarsonic acids with trypsin is under further study.

Effect of Phenylarsonic Acids on SubtilisinsThe four phenyl- arsonates, examined above with cY-chymotrypsin, showed a time- dependent inhibition of the subtilisins at pH 6.0. Here also, in all cases, an apparent zero order rate of substrate hydrolysis was attained when the assays were allowed to proceed for a sufficient period of time (see Fig. 8). In contrast to the results

01 I 1 I 1 1 I 0 4 8 12 16 20 24 28

TIME (MINUTES)

FIG. 8. Kinetics of hydrolysis of benzoyl-L-arginine ethyl ester (0.025 M) by Carlsberg subtilisin (0.074 mg per ml) in 0.1 M CaCl2 at pH 6.0 and 37”. Curve 1, no inhibitor; Curve 2, in the presence of 4 X 10-a M phenylarsonate. - - -, extension of the apparent zero order rate reached after 12 min in the presence of the inhibitor.

2

3 3 4

2 l&k&! 5

6

0 0 2 4 6 8

TIME (MINUTES)

-0 2 4 6

Cp-ARSANILATEI M x IO3

FIG. 9. Determination of the dissociation constant for the Novo subtilisin-p-arsanilate complex. Mixtures of Novo sub- tilisin (3.15 X 1W M) and p-arsanilate at the concentrations 1, 0; 2, 6.6 X 10-4 M; S,l.32 X lo- M; 4,1.67 X IO-$ M; 5, 3.33 X lo- M; and 6, 6.66 X 10-s M were incubated for 240 min in 0.2 M acetate- 0.02 M CaCls buffer at, pH 6.0 and 25”. This period exceeded that required for the attainment of equilibrium. The extent of inhibi- tion was determined by assaying the initial rate of esterolysis of N-benzoyl-L-arginine ethyl ester. Assays were performed by addition of 50 ~1 of incubation mixture to 5 ml of 0.025 M substrate in 0.1 M CaClz at pH 6.0 and 25”. A, initial rates of substrate hydrolysis obtained with the incubation mixtures listed above. B, percentage inhibition under the above conditions as a function of p-arsanilate concentration; the curve drawn is theoretical as calculated for a Kd isa o f 1.70 X 1W M for a 1:1 enzyme-inhibitor complex.

TABLE II

Dissociation constants of complexes of subtilisins with various phenylarsonates

All incubations were performed at pH 6.0 in 0.2 M acetate-O.02 M

Cal& buffer at 25”. Titrimetric determination of KI values was performed as described in the legend to Fig. 9.

I 103 x Kr

Inhibitor Titrimetric determination Dye

Carlsberg displacemenf, b

subtilisin’” Nova subtilisina Now subtihn

M M M

p-Nitrophenylarsonate... 9.7 f 0.5 12.1 f 2.0 9.8 f 0.9 p-Tolylarsonate. 2.3 i 0.3 4.5 f 0.5 8.1 f 0.7 p-Arsanilate. 1.1 f 0.2 1.7 f 0.1 1.8 f 0.1 Phenylarsonate. 1.0 i 0.1 1.3 f 0.2 0.68 + 0.1

u Mean of eight determinations at different inhibitor concentra- tions.

b Determined after 5 hours of incubation at 25”. The details of experimental conditions are given in the legend to Fig. 10. The calculations were based on EM at 390 mp for 4-(4’-aminophenylazo)- phenylarsonate of 17,800 (l), a Kdiaa value for the Novo subtilisin- dye complex of 6.1 X 10e6 M, and a ARM value for the complex of 9,730 at 478 rnp in 0.2 M acetate-O.02 M CaClz buffer, containing 3.7y0 ethanol, at pH 6.0. The values given are the mean of deter- minations at two different protein concentrations.

obtained with p-nitrophenylarsonate and cr-chymotrypsin, none of the inhibitors tested with the subtilisins exhibited its inhibitory effect within the time of mixing. Further, the rate and extent

of inhibition at a fixed inhibitor and enzyme concentration in

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the assay mixture were independent of substrate concentration over the range, 4 X 10e4 M to 2.5 X 1O-2 M, for N-benzoyl-n- arginine ethyl ester. Similar results had been obtained earlier for the inhibition of the subtilisins by 4-(4’-aminophenylazo)- phenylarsonate (1). Hence, in sharp contrast to the results obtained with a-chymotrypsin, the initial site of binding of the phenylarsonates on the subtilisins is distinct from the substrate- binding site.

Determination of Kr Values by Titration--As described above for the cY-chymotrypain-p-arsanilate complex and, earlier, for the subtilisinl-(4’-aminophenylazo)phenylarsonate complex (l), the dissociation of the subtilisin-phenylarsonate complexes on dilution at pH 6 and 25” was sufficiently slow to permit esti- mation of the amount of free enzyme present in the incubation mixtures by assaying at pH 6 and measuring the initial rate of esterolysis. A representative experiment leading to the deter- mination of the KI value for the subtilisin-p-arsanilate complex is presented in Fig. 9. The KI values calculated from such data are given in Table II.

I

360 420 480 540

WAVELENGTH (mp)

FIG. 10. Displacement of 4-(4’-aminophenylazo)phenylarsonate from Novo subtilisin by phenylarsonatee: difference spectra (AA) obt,ained by comparing the absorbance of a mixture of 4-(4’- aminophenylazo)phenylarsonate (6.6 X 1W5 M) and Novo sub- tilisin (2.22 X lo-* M) with that of the dye alone, in the presence of Curve 1, no inhibitor; Curve 8, p-nitrophenylarsonate (4.4 X 10d3 M) ; Curve 3, p-tolylarsonate (4.4 X 10e3 M) ; Curve 4, p-arsan- ilate (4.4 X 10-a M) ; and Curve 5, phenylarsonate (1.1 X 10-a M). The spectra were obtained after the mixtures were incubated for 300 min at 24” in 0.2 M acetate-O.02 M Calls at pH 6.0. In each case, the reference dye solution contained an amount of the ap- propriate inhibitor matching that in the incubation mixture. The light path was 1 cm.

Or 6.0 6.5 ZO 7.5 8.0 8.5

PH

FIG. 11. pH dependence of the inhibition of Novo subtilisin by phenylarsonate. The degree of inhibition was determined by assaying the esteratic activity toward N-benzoyl-L-arginine ethyl ester (0.025 M) in 0.1 M CaCls at 25”, as a function of pH, at an enzyme concentration of 0.04 mg per ml in the presence and in the absence of 1.9 X IO-+ M phenylarsonate. Sufficient time was allowed in assays carried out in the presence of inhibitor to attain apparent zero order rates of substrate hydrolysis (see text). The curve drawn is theoretical, as calculated for the protonation of a single group of pK’, 7.17.

Determination of KI Values by Displacement of .&&‘-Amino- phenylazo)phenylarsonic Acid-It was shown earlier that 4-(4’- aminophenylazo)phenylarsonate bound to the subtilisins at a single site yields an inactive enzyme-dye complex (1). Phenyl- arsonates were found to compete for this binding site (see Fig. 10). Since the Kdiss of the protein-dye complex could be readily determined (I), the affinity of the various phenylarsonates for the subtilisins could be determined by dye displacement just as described above for the proflavine-chymotrypsin system. The experimental conditions used are given in the legend to Fig. 10, and K, values obtained from such measurements are listed in Table II.

The order of affinities of the various inhibitors for Novo sub- tilisin was the same as that obtained by the titrimetric procedure. The Kr values obtained by both procedures for p-nitrophenyl- arsonate and p-arsanilate were in good agreement. The con- stants obtained by the two methods for p-tolylarsonate differed by a factor of 2. This discrepancy may be due to the presence of 3.7% ethanol in the incubation mixtures used in the deter- mination of dye displacement.

pH Dependence of Inhibition of Subtilisins by Phenylarsonates

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3700 Inhibition of “Xerine” E&erases by Phenylarsonates Vol. 243, No. 13

The inhibition of the subtilisins was studied as a function of pH with N-beazoyl-L-argiaille ethyl ester as substrate. At each pH value, in assays performed in the presence of the inhib- itor, substrate hydrolysis was followed until the time-dependent change in enzyme activity was complete and an apparent zero order rate of substrate hydrolysis was attained (see Fig. 8, for example). The percentage inhibition at each pH value was determined from the ratio of this rate to that observed in the absence of inhibitor. It may be added that the K, determined, at fixed inhibitor concentration and pH value, from the apparent zero order rates obtained at different substrate concentrations was the same as that of the noninhibited enzyme, indicating that only fully active enzyme and totally inactive ET complex were present. Since the K, of the subtilisins for N-benzoyl-L- arginine ethyl ester does not vary significantly with pH over the range, 6 to 8.5 (5), the percentage inhibition values deter- mined at constant enzyme, inhibitor, and substrate concen- trations represent a direct measure of the K1 as a function of pH. The data obtained wit.h phenylarsonate and Novo subtilisin are shown in Fig. 11. It is striking that, just as with a-chymo- trypsin, the results can be fitted closely by a theoretical curve for the ionization of a single group wit.h a pK’, value of 7.17.

DISCUSSION

The results presented above show that phenylarsonates are powerful inhibitors of chymotrypsin, trypsin, and the subtilisins. The primary structures of these two classes of enzymes exhibit profound differences; they do share, however, a very similar mechanism of action (5, 24). The observed similarity in their behavior toward the phenylarsonates is clearly related to the characteristics of the catalytic sites of these enzymes. The above proteins are all members of the group of serine esterases (25). Enzymes classified as belonging to this group are all specifically and stoichiometrically inhibited by certain organic phosphorylating agents, in particular by diisopropyl fluoro- phosphate. The inhibition depends on the conversion of a specific serine residue essential to the catalytic activity of these enzymes into the diisopropylphosphoryl derivative (25). Scat- tered reports have appeared in the literature indicating that other members of the serine esterase group are inhibited by pentavalent arsenicals. Equine acetylcholinesterase (26), liver esterase from several sources (27, 28), and pancreatic aliesterase

SrEP 1

I HC =F-CH,-fH

;H2-OH HO/&- +,I, 0

+c,NH f0

-NH-CH-CO- H

S?-EP 2

AS -0'4 'O-

0 -NH-CH-CO- H

FIG. 12. A possible scheme for the inhibition of chymotrypsin and srtbtilisins by phenylarsonates.

(29) are all inhibited by p-arsanilate as well as by diisopropyl fluorophosphate. From the meager data available in the litera- ture, t.he characteristics of the inhibition observed in these cases appear consistent with those described in the present study. A tent,ative conclusion may thus be reached that inhibition by pentavalent arsenicals is a general property of the serine esterases.

The kinetics of the inhibition by the phenylarsonates shows a profound dependence on the specific inhibitor and enzyme under study. This is most strikingly illustrated for chymotrypsin by comparing the instantaneous competitive inhibition observed with p-nitrophenylarsonate and the very slow inhibition ob- served with p-arsanilate. Subtilisin, in contrast, interacts slowly with both of the aforementioned inhibitors. The impor- tance of the structure of the phenylarsonate for its effectiveness as an inhibitor of a particular enzyme was most vividly illustrated in a preceding study with 4-(4’.aminophenylazo)phenylarsonate (I). This strong inhibitor of the subtilisins failed to interact with either trypsin or chymotrypsin (I). The relative affinity of the four inhibitors examined in this study toward cr-chymo- trypsin was p-arsanilate > p-tolylarsonate > p-nitrophenyl- arsonate > phenylarsonate, whereas the order with respect to Novo and Carlsberg subtilisins was phenylarsonate > p-arsanil- ate > p-tolylarsonate > p-nitrophenylarsonate. Clearly, the structure of the inhibitor influences its affinity for the enzyme. The above differences in the order of binding affinity for sub- tilisins and chymotrypsin may be viewed as reflecting differences in the details of the geometry of the inhibitor-binding sites.

Both oc-chymotrypsin and the subtilisins show a time-depend- ent interaction with the phenylarsonates. With the subtilisins, the presence of substrate, even at a hundred-fold molar excess over the inhibitor, is without effect on the rate and extent of inhibition. The K, values for the substrates examined are within an order of magnitude of the observed K, values, and some influence on the rate of inhibition would be expected. The results suggest that the arsenicals interact at a site distinct from the substrate-binding site and that the initial complex formed is still catalytically active, and that it then undergoes a slow rear- rangement to give an inactive enzyme.

fast slow E+I e (EI)active e (EI’)tnactive

In the case of ar-chymotrypsin, the first step appears to involve interaction at the substrate- and competitive inhibitor-binding site, and hence both the rate and the extent of inhibition are dependent on substrate as well as inhibitor concentration.

The pH dependence of the catalytic rate constant for chymo- trypsin (30) and for subtilisins (5), for a variety of substrates, is associated with a sigmoid curve characterized by a pK’ of 6.5 to 7.3. This has generally been interpreted to represent the involvement of an un-ionized histidine residue in the catalysis. It is striking that the inhibition of both a-chymotrypsin and subtilisins by phenylarsonates exhibits exactly the reverse type of pH dependence, adhering closely to the theoretical curve for the protonation of a group of pK’ 7.15 (Figs. 6 and 11). The simplest interpretation of this observation is that the binding of the inhibitor requires the presence of a protonated histidine residue at the active site of these enzymes. The phenylarsonic acids used in this study have the following ionization constants (pKal and pK& in aqueous solution at 25” (20): p-amino-, 2.0 and 8.92; p-tolyl-, 3.70 and 8.68; unsubstituted, 3.47 and 8.48; and p-nitro-, 2.90 and 7.80. At pH 6, the phenylarsonic acids

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exist as the monovalent anions, at pH values above 7, as mixtures of the mono- and divalent anions. Since no correlation between the pK,, value and binding affinity was observed, and since the pH dependence of the inhibition appears to be independent of the pK,, of the inhibitor (see Fig. 6), it would appear that the mono- and divalent anions have a similar affinity for the binding site of the enzymes.

A possible scheme for the interaction of the phenylarsonates with chymotrypsin and the subtilisins is presented in Fig. 12. The initial interaction (Fig. 12, Step 1) of the inhibitor with the enzyme is envisaged as depending on an electrostatic interaction between the phenylarsonate anion and a protonated histidine residue, as well as an apolar interaction involving the aromatic ring. Subsequently, ester formation may take place with the serine residue at the active site (Step 2). This ester may be formed as a consequence of lowered activity of water at the bind- ing site and stabilized by the interactions mentioned above. Step 2 is suggested on the basis of the following lines of reasoning. This reaction is analogous to that established by I*0 exchange to occur between a-chymotrypsin and carboxylic acids such as N- acetyl.L-tryptophan and benzoyl-L-phenylalanine.1 The ac- chymotrypsin-p-arsanilate complex exhibits considerable stabil- ity in concentrated guanidine solutions-a property possessed to a varying extent by acyl-chymotrypsins (23). The observed lability of the complex in alkaline solution is also in keeping with this formulation. This mechanism is amenable to test by I80 exchange, and such studies are envisaged for the future. The alternative interpretation is that the binding of phenylarsonates results in considerable stabilization of the native structure with- out any covalent bond formation. The dramatic stabilization toward denat.uring agents of bovine pancreatic ribonuclease, through the strong binding of a single phosphate anion, may be mentioned in this context (31-33). The observed slow inactiva- tion by the phenylarsonates might then represent a local time- dependent conformational rearrangement within a rapidly formed enzyme-inhibitor complex of low stability. Further studies to examine the influence of phenylarsonates on the structure of chymotrypsin and the subtilisins will be undertaken.

Finally, the phenylarsonates may have some practical value as reversible inhibitors of serine esterases, both in purification procedures, in which it is necessary to keep autodigestion at a minimum, and as a means of suppressing proteolytic activity when other ways of achieving this are undesirable.

Acknowledgments-I would like to thank Miss Carol for excellent technical assistance. I am also indebted Emil L. Smith for his interest in this work and many discussions.

1 See p. 218 of Reference 30 for a review.

Sander to Dr. helpful

1. 2.

3. 4.

5. 6.

7.

8.

9.

10.

11. 12.

13.

14. 15. 16. 17.

18.

19.

20.

21. 22. 23.

24.

25. 26,

27.

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A. N. GlazerAND THE SUBTILISINS

-CHYMOTRYPSINαInhibition of "Serine" Esterases by Phenylarsonic Acids:

1968, 243:3693-3701.J. Biol. Chem. 

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