Studies on the Mechanism and Active Site for the Esterolytic Activity

THE JOIJRNAL OF Bm~oarcn~ CHEMISTRY Vol. 239, No. 7. July 1964

Printed in U.S.A.

Studies on the Mechanism and Active Site for the Esterolytic Activity of 3-Phosphoglyceraldehyde Dehydrogenase”

ERIK J. OLsoNt AND JANE HARTING PARKS

From the Department of Physiology, Vanderbilt University School of Medicine, Nashville 5, Tennessee

(Received for publication, January 23, 1964)

It has been found that the 3-phosphoglyceraldehyde dehydrogenase, which is crystallized from rabbit muscle or yeast, can catalyze the hydrolysis of p-nitrophenyl acetate (1, 2). The inhibition of this hydrolysis by the participants of the dehydrogenase reaction, namely, 3-phosphoglyceraldehyde, glyceraldehyde, nicotinamide adenine dinucleotide, phosphate, and arsenate, indicated that the catalytic site for the hydrolytic reaction involved at least a portion of the site for the normal oxidation activity. The isolation of an acetyl-enzyme complex, which was prepared with l4C-p-nitrophenyl acetate and the dehydrogenase, showed that there were three or, possibly, four active sites in the enzyme. A sulfhydryl group was implicated at the catalytic site, since both the formation of the acetyl-enzyme complex and the over-all hydrolysis of p-nitrophenyl acetate were inhibited by iodoacetic acid (1).

The involvement of the cysteine moiety was definitely established by the isolation of 14C-acetyl peptides and studies on the amino acid sequence of the active site (3,4). It was shown that the three or four active sites were identical, and the r4C-acetyl group was bound in thioester linkage to a cysteine moiety in the following sequence (3).

Lys-Ileu-Val-Ser-AspNHrAla-Ser-CyW4COCH3 (Thr, Thr, AspNHz)

An S-carboxymethyl peptide from an enzyme labeled with 14C- iodoacetic acid extended the sequence and proved that the iodoacetic acid reacted selectively with only one of the two cysteines in the heptadecapeptide (3).

Ileu-Val-Ser-AspNH*-Ala-Ser-CySl%!H&OOH- Thr-Thr-AspNH&yS-Leu-Ala-Pro-Leu-Ala-Lys

In the present paper the esterolytic activity of the dehydrogenase was further investigated in order to examine the general scope of the esterolytic reaction and the mechanism of the catalysis. The specificity of the enzyme was tested with different phenyl acetate derivatives and various alkyl esters, including N-acetyltyrosine ethyl ester and L-tyrosine ethyl ester. Since cysteine and glutathione can accelerate the breakdown of p-nitrophenyl acetate (5), it was of interest to compare the dehydrogenase, cysteine, and glutathione with regard to their

* This work was supported by grants from the Muscular Dys- trophy Association of America, the National Science Foundation, and the United States Public Health Service.

t Postdoctoral fellow of the Muscular Dystrophy Association of America.

$ Career Development Awardee of the United States Public Health Service.

ability to facilitate the cleavage of the various phenyl acetate derivatives. The results with the dehydrogenase are discussed in relation to other esterolytic enzymes such as chymotrypsin (6) and wheat germ lipase (7). The esterolytic reaction is briefly contrasted with the so-called phosphatase activity of the dehydrogenase, which involves the hydrolysis of acyl pos- phates (8).

EXPERIMENTAL PROCEDURE

Preparation of Phenyl Acetate Derivates-p-Nitrophenyl acetak was prepared as previously described by Balls and Wood (9). The melting point of this material was 78”, as compared to the previously reported melting point of 80-81” (9). Both o- and m-nitrophenyl acetate were synthesized in essentially the same manner as p-nitrophenyl acetate. However, a dense oily layer separated when the ice-cold 2% aqueous acetic acid was added after reaction of the nitrophenols and acetic anhydride in pyridine. This layer was washed twice with 3 volumes of 2% aqueous acetic acid. Both o- and m-nitrophenyl acetate were crystallized by dissolving the washed material in ethanol and adding 2 volumes of water. The melting points of the synthesized o- and m-nitrophenyl acetates were 37” and 54”, respectively, as compared to the previously reported literature values of 41” (10) and 55-56” (11). The spectrophotometric analysis of o-nitrophenol liberated from the o-nitrophenyl acetate by alkaline hydrolysis indicated that this ester was 99% o-nitrophenyl acetate.

The procedure of Balls and Wood was also used to synthesize d,&Uromophenyl acetate. The melting point of this preparation was 36”, which was in agreement with the previously reported melting point for this compound (12).

p-Methoxyphenyl acetate was synthesized by a modification of the procedure for p-nitrophenyl acetate used by Balls and Wood (9). The reaction mixture, containing p-methoxyphenol, acetic anhydride, and pyridine, was allowed to stand for 1 day instead of 4 hours. A dense amber oil, which formed on the addition of 2% acetic acid, was separated off and then taken up in an equal volume of ethanol. Upon the addition of 10 volumes of water at O-4”, the oil formed again. This procedure with ethanol and water was repeated until the p-methoxyphenyl acetate crystallized. The melting point of this material was 30-31”, while the value reported in the literature is 31-32” (13).

The procedure of Balls and Wood (9) was also modified for synthesizing p-chlorophenyl acei&. The reaction mixture was allowed to stand for 3 or 4 days at room temperature. The oily layer, which was formed on the addition of 3 volumes of ice-cold 2% aqueous acetic acid, was washed four times with 2 volumes

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July 1964 E. J. Olson and J. H. Park 2317

of 2% aqueous acetic acid. As with the p-methoxyphenyl acetate, the oily layer was washed several times by mixing it with 3 volumes of ethanol and separating it out by the addition of an excess of water. This oil was then dried over calcium chloride in a vacuum. When cooled to -2O”, it solidified. The melting point of this material was 8” as compared to the previously reported value of 7-8” (14).

o-Chlorophenyl acetate was synthesized by a modification of the method of Wohlleben (14). Crude o-chlorophenyl acetate was collected by adding an excess of cold water to the reaction mixture at 0” and purified by vacuum distillation. The melting point of this preparation coincided with the reported melting point of -20” (14).

The procedure of Balls and Wood (9) was further modified for the synthesis of 2-chloro-Qdrophenyl acetate. The phenol (7 g) was allowed to stand overnight at room temperature in 65 ml of pyridine and 6.5 ml of acetic anhydride. After addition of acetic acid, the crystals were collected and washed with 2‘% acetic acid. These crystals were dissolved in toluene and shaken three times with + volume of 1 N sodium carbonate. The toluene solution was concentrated by heating at approximately 50” and placed at O-4” overnight for crystallization. The melting point was 61-62” as compared to the reported value of 63” (15).

To synthesize 4-chloro-2-nitrophenyl acetate, the reaction mixture of Balls and Wood (9) was altered. 4-Chloro-2-nitrophenol (14 g) was dissolved in 60 ml of pyridine and 12 ml of acetic anhydride and allowed to stand overnight. When the 2% acetic acid was added, the crude 4-chloro-2-nitrophenyl acetate separated as a dense oily layer. This dark oily layer was taken up in 75 ml of toluene and separated from the aqueous layer. The aqueous layer was shaken with 35 ml of toluene, which was then combined with the first toluene extraction. The combined toluene layers were washed twice with one-third the volume of 2% aqueous acetic acid at O-4” and three times with two-thirds of the volume of 1 N sodium acetate. The toluene layer was then concentrat,ed to 20 ml. 2,2,4-Trimethylpentane (40 ml) was then added and the solution was placed at 0” overnight. Yellow crystals of the unreacted phenol and dark brown crystals of 4-chloro-2nitrophenyl acetate were separated from each other mechanically. The partially purified crystals were suspended in 2,2,4-trimethylpentane and heated to 40-50”. A dense vitreous oil formed, which could be separated from the lighter layer of 2,2,4-trimethylpentane. This oil crystallized on cooling. The resulting crystals were taken up in a minimal amount of toluene and recrystallized by the addition of 2 volumes of 2,2,4trimethylpentane at 0”. The melting point was 47-49” as compared to the previously reported temperature of 47-48”

(16). Ethyl p-nitrophmyl carbonate was synthesized by the method of

Ransom (17). This substance was recrystallized in ethanol to a melting point of 66”. The reported value is 67-68” (17).

The method of Ransom was also used to synthesize ethyl 4- chloro-2-nitrophenyl carbonate and ethyl 2-chloro-4-nitrophenyl carbonate. The crystalline ethyl 4-chloro-2nitrophenyl carbonate melted at 59-60”. This value agreed with the previously reported value of 60” (16). Ethyl 2-chloro-4-nitrophenyl carbonate melted at exactly 45”. Optical density readings at 400 mp after alkaline hydrolysis indicated that the ethyl 2-chloro-4- nitrophenyl carbonate preparation was 98 $Zo pure.

p-Nitroacetanilide was synthesized by adding 2 g of p-nitro- aniline to 100 ml of acetyl chloride and 100 ml of toluene and

heating for 4 hours at 60”. The reaction mixture was cooled to room temperature and filtered. The precipitate was dissolved in hot toluene, cooled, and separated by filtration. The solid was redissolved in 30 ml of acetone. The precipitate, which ap- peared when 50 ml of water were added, was removed by filtration. When the filtrate stood at O-4”, white needles of p-nitroacetanilide were formed. The melting point was 2X-216”, as previously reported (18).

Ethyl p-nitrobenzoate was synthesized by refluxing in ethanol and sulfuric acid. After recrystallization from ethanol and water, the melting point was 55”. The reported melting point was 57” (19). Methyl p-nitrobenzoate was synthesized by the same method. The crystals were dissolved in toluene and shaken briefly with 1 N sodium carbonate. An equal volume of 2,2,4- trimethylpentane was then added to the toluene layer. Crystals of methyl p-nitrobenzoate formed when this solution was allowed to stand overnight at 04”. The melting point of 96” coincided with that reported for this compound (19).

Materials and Enzyme Preparation-L-Tyrosine ethyl ester, n-cysteine hydrochloride, and monosodium glutathione were obtained from Nutritional Biochemicals Corporation. p-Nitro- phenyl phosphate was a product of the California Corporation for Biochemical Research, and N-acetyl-n-tyrosine ethyl ester was a gift from L. W. Cunningham. Phenyl acetate was obtained from Eastman Organic Chemicals Department, o-iodosobenzoate from the Sigma Chemical Company, and 3-phosphoglyceraldehyde from Calbiochem.

3-Phosphoglyceraldehyde dehydrogenase was prepared from rabbit muscle by the method of Cori, Slein, and Cori (20). The enzyme was recrystallized twice in the presence of 0.001 M Ver- sene. Enzyme-bound NAD was removed with activated charcoal by the method previously described (21). The ratio of the optical density readings at 280 and 260 rnp was 1.9, indicating that the NAD was completely removed. The NAD-free enzyme was dialyzed against Tris-Versene buffer to eliminate ammonium sulfate, which causes appreciable hydrolysis of some of the substrates. For the calculations in this paper, the values used for the molecular weight and the extinction coefficient are those suggested by Fox and Dandliker (22).

Methods-The hydrolysis of the substrates was followed either titrimetrically with a Radiometer type TTTl instrument, which recorded the acid production from the liberated acetate and phenol at constant pH, or spectrophotometrically by measuring the increase in absorption due to the liberated phenol. The results obt,ained by the two methods were in agreement..

When the enzymatic reactions were followed with the titrim- eter, the pH of the solution was adjusted by adding 0.00955 N

NaOH to the solution, which usually contained the 1.6 mg of enzyme in a volume of 6 ml. The reaction was started by adding approximately 11 pmoles of substrate. The base consumption that occurred during the enzymatic reaction was corrected for spontaneous hydrolysis of the substrates. The controls were run immediately before and after the enzyme was tested. In all cases the results obtained titrimetrically were corrected for the ionization of the liberated phenolic group. For these calculations the following pK values obtained by Judson and Kil- patrick (23) were used: o-nitrophenol, 7.2; p-nitrophenol, 7.1; m-nitrophenol, 8.3; o-chlorophenol, 8.4; and p-chlorophenol, 9.3. The pK values of 5.3 for 2-chloro-4-nitrophenol, 5.9 for 4-chloro- 2-nitrophenol, and 7.7 for 2,4-dibromophenol were determined experimentally by the authors from pH-titration curves.


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Esterolytic Activity of S-Phosphoglyceraldehyde Dehydrogenase Vol. 239, No. 7

TABLE I Maximal velocities of hydrolysis of phenyl acetate derivatives

catalyzed by NAD-free 9-phosphoglyceraldehyde dehydrogenase The maximal velocities of the hydrolysis of the various deriva-

tives were obtained with a charcoal-treated, NAD-free enzyme which was dialyzed for 4 hours against 0.005 M Tris and 0.001 M

Versene buffer at pH 7.0. Varying amounts of enzyme and substrates were used depending on the velocity of the reaction and the solubility of the substrate. For example, the V,,, forp-nitrophenyl acetate was obtained with 1.2 mg of substrate and 1.0 mg of enzyme in a total volume of 3.0 ml. In the calculations of the Lineweaver-Burk plots for 2-chloro-4-nitrophenyl acetate and 4-nitro-2-chlorophenyl acetate, the highest substrate concentration was 0.4 mg, and the enzyme concentration, 1.0 mg. For the spectrophotometric measurements, the pH was main- tained at pH 8.6 with 40 pmoles of Verona1 buffer in a total volume of 3 ml. In the titrimetric work the pH was automatically regu- lated at 8.6 with a Radiometer type TTTl instrument as described in “Experimental Procedure.” The reactions were carried out at room temperature.

Substrate

2-Chloro-4-nitrophenyl acetate 4-Chloro-2-nitrophenyl acetate p-Nitrophenyl acetate

o-Nitrophenyl acetate 2,4-Dibromophenyl acetate m-Nitrophenyl acetate o-Chlorophenyl acetate p-Chlorophenyl acetate Phenyl acetate p-Methoxyphenyl acetate

VIII,,

umoles/min/mg eneynte

0.11*t o.o95*t 0.115* O.llO$ 0.090* o.o55*t 0.0571 0.040Q 0.022$

O$ O$

pK of phNl0l

5.3 5.9 7.1

7.2 7.7 8.3 8.4 9.3

10.0 10.2

* Data obtained spectrophotometrically. t Value based on Lineweaver-Burk plot (24). $ Data obtained titrimetrically.

Spectrophotometrically the hydrolysis of p-nitrophenyl acetate and 2-chloro-4-nitrophenyl acetate was followed at 400 rnp, o-nitrophenyl acetate at 420 rnp, 4-chloro-2-nitrophenyl acetate at 430 rnp, and 2,4-dibromophenyl acetate at 300 ml.c. When the reactions were followed spectrophotometrically, they were generally started by the addition of the enzyme. Corrections were made for the spontaneous hydrolysis of the compound under the same experimental conditions.

RESULTS

Spec$icity and Properties of NAD-free Dehydrogenase as Catalyst of Esterolytic Reactions-The maximal velocities for the enzymatic hydrolysis of a number of aryl esters are shown in Table I. These values were obtained either from the spectral changes arising from the liberation of a given phenol or from the titrimetric measurement of the acid produced by the hydrolytic reaction. The data presented for p-nitrophenyl acetate demonstrated that both methods were in substantial agreement. Because of the insolubility of the halogenated derivatives, the maximal rates derived by extrapolation from Lineweaver-Burk plots are subject to greater error then those for the more soluble compounds, for which rates close to the maximal value could be realized experimentally. In the latter case, the highest observed

value has been taken as a sufficiently accurate expression of V Inax. It is apparent that generally the maximal velocity of the hydrolysis decreased as the pK of the corresponding phenol increased. However, changes in the pK below 7.2 did not affect the rate of hydrolysis as shown by the comparison of p-nitrophenyl acetate and 2-chloro-4-nitrophenyl acetate. No hydrolysis of phenyl acetate or p-methoxyphenyl acetate could be detected between pH 7 and 9, even at high substrate (14 pmoles) and enzyme (3.7 mg) concentrations.

The effect of pH on the maximal velocities of the hydrolysis of five of the phenyl acetate derivatives is presented in Fig. 1. There was no substantial difference in the pH curves obtained spectrophotometrically and titrimetrically for p-nitrophenyl acetate. No attempts were made to extend the curves beyond pH 8.9 because of the instability of the enzyme (20) and the increased spontaneous breakdown of the substrates at the higher pH values. As the pH was raised from 8.0 to 8.6, the maximal rate of hydrolysis increased a-fold for p-nitrophenyl acetate and 2-chloro-4-nitrophenyl acetate, a-fold for m-nitrophenyl acetate, and Id-fold for p-chlorophenyl acetate. Since the curves for p-nitrophenyl acetate and the chloronitrophenyl esters were similar, it was considered that these hydrolyses catalyzed by the NAD-free dehydrogenase might be subject to a common rate- limiting factor. The possibility of product inhibition of 2-chloro- 4-nitrophenyl acetate hydrolysis was checked titrimetrically by

7.5 0.0 8.3 6.6 8.9

PH

FIG. 1. The effect of pH on the maximal velocities of the hydrolysis of phenyl acetate derivatives catalyzed by the NAD-free 3-phosphoglyceraldehyde dehydrogenase. The reaction mixture for m- and p-nitrophenyl acetate contained 11.5 pmoles of substrate, which were added in 0.2 ml of 50c$ acetone, and 1.6 mg (0.011 pmole) of NAD-free dehydrogenase. Total volume was 6 ml. The reaction mixture for p-chlorophenyl acetate contained 106 pmoles of substrate and 5.4 mg (0.038 @mole) of enzyme. Total volume was 18 ml. The reactions were followed in the Radiom- eter titrator, model TTTl, at room temperature. The reaction mixture for the chloronitro derivatives contained 1.0 mg (0.007 pmole) of enzyme, 40 pmoles of Verona1 buffer at the designated pH, and varying amounts of the substrate in 0.2 ml of 50yo ethanol. The reaction was measured in a Beckman spectrophotometer at room temperature. Total volume was 3.0 ml.


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preincubating the enzyme with 6 x lop3 M 2-chloro-4-i&o- phenol, which represented a 2500-fold excess over the enzyme on a molar basis. At pH 8.0 there was no inhibition, and at pH 8.6, only 10%. Likewise, p-chlorophenol did not effect the enzymatic rate of hydrolysis of p-chlorophenyl acetate.

In Table II it can be seen that the aryl esters of ethyl carbonate are hydrolyzed less rapidly by the NAD-free dehydrogenase than the corresponding acetyl derivatives. Although the maximal rate of hydrolysis of ethyl 2-chloro-4-nitrophenyl carbonate approaches that of 2-chloro-4-nitrophenyl acetate, the maximal rates of hydrolysis of the other nitrophenyl acetate derivatives are several times more rapid than the corresponding rates for the ethyl carbonate esters. As expected for the cleavage of the aryl ester bond at pH 8.6, about 2 equivalents of acid are produced per mole of ethyl p-nitrophenyl carbonate cleaved. As with the nitrophenyl acetate derivatives, the rate of hydrolysis of the carbonate derivatives rose sharply as the pH was increased. The relative increases were approximately 2.5-fold for ethyl p-nitrophenyl carbonate and ethyl 2-chloro-4-nitrophenyl carbonate as the pH was increased from pH 8.0 to 8.6.

Both p-nitrophenyl phosphate and p-nitroacetanilide were found to be completely resistant to hydrolysis by the dehydrogenase at pH 7.5 and 8.6. Because of its insolubility, only 1 pmole of p-nitroacetanilide was tested in 3 ml of 8% acetone with 5.0 mg of enzyme. Under comparable conditions the enzyme liberated enough p-nitrophenol from p-nitrophenyl acetate to increase the optical density readings at 400 rnp at the rate of 0.280 per minute at pH 7.5 and 0.600 per minute at 8.6. At pH 7.5 and 8.6, 3 pmoles of p-nitrophenyl phosphate were mixed with 5 mg of enzyme without any discernible hydrolysis.

r.,-Tyrosine ethyl ester and N-acetyltyrosine ethyl ester, which are commonly used as substrates for chymotrypsin, were inactive as substrates for the NAD-free dehydrogenase. L-Tyrosine ethyl ester (6 pmoles) and N-acetyltyrosine ethyl ester (10 pmoles) were tested in a pH range from 7 to 9 with approximately 4 mg of enzyme. Under these conditions at pH 8.0, chymotrypsin would have hydrolyzed half of the N-acetyltyrosine ethyl ester in 0.1 second (25).

Ethyl p-nitrobenzoate, methyl p-nitrobenzoate, ethyl acetate, and isopropenyl acetate were found to be inactive as substrates in a pH range from 7 to 9. The dehydrogenase (4 mg) was tested with 4 pmoles of ethyl and methyl p-nitrobenzoate in a total volume of 6 ml. Although the solubility of ethyl p-nitrobenzoate prevented the use of higher substrate levels, there was no detecta- ble hydrolysis of this ester even in the presence of 10 mg of enzyme. Ethyl acetate (10 wmoles) and isopropenyl acetate (9 pmoles) were not hydrolyzed by 4 mg of enzyme.

The relationship between the rates of the enzymatic hydrolysis and the spontaneous hydrolysis of nitrophenyl esters is summa- rized in Table III. The measured rates of the enzymatic hydrolysis and the spontaneous breakdown of p-nitrophenyl acetate were 0.088 and 0.012 pmole per minute, respectively. In the case of both the enzymatic and the spontaneous hydrolysis, the rate for p-nitrophenyl acetate was arbitrarily set at lOO%, and the other derivatives were related to p-nitrophenyl acetate. There is close correlation between the comparative rates for the catalytic hydrolysis and the lability of the various derivatives. The enzymatic rates in the table were determined at subsaturation levels of substrates under conditions suitable for this comparative rate study. Therefore, the relative rates for the first four compounds show variations which are not observable in the

V,,, values in Table I. These variations are roughly in line with the pK, values of the phenols listed in Table I.

Cleavage of Aryl Esters by Model Systems-Cysteine and

TABLE II Comparison of rates of hydrolysis of acetyl and ethyl carbonyl

aryl esters

The reaction mixture contained 1.0 mg (0.007 pmole) of the NAD-free dehydrogenase, 40 pmoles of Verona1 buffer, pH 8.6, and different amounts of the substrates in 0.2 ml of 50% ethanol. The reactions were followed spectrophotometrically at room temperature. Appropriate blanks were run to correct for the spontaneous hydrolysis of the substrates. The maximal rates of hydrolysis of the ethyl nitrophenyl carbonate derivatives were determined from Lineweaver-Burk plots.

SUBSTRATE

NO2

f No2f---3- OCCH,

0 N02aOtOCH2CH3

Vmox

IO-’ molcs/min/mg enzyme

I.1

0.73

0.95

0. 22

I. I

0. I

TABLE III

Comparison of enzymatic and spontaneous rates of hydrolysis of nitrophenyl derivatives

The enzymatic hydrolysis was carried out with 1.0 mg (0.007 rmole) of enzyme and 2.0 pmoles of substrate in 3.0 ml of 0.014 M Verona1 buffer, pH 8.6. This concentration of the derivatives was at subsaturation level and the compared rates were not V,,, values. The spontaneous hydrolysis of 2 pmoles of the various esters was also followed spectrophotometrically at pH 8.6.

Ester

Rate of hydrolysis

Enzymatic Spontaneous

2-Chloro-4-nitrophenyl acetate .......... 4-Chloro-2-nitrophenyl acetate. ......... p-Nitrophenyl acetate. ................. o-Nitrophenyl acetate ................... m-Nitrophenyl acetate. ................. Ethyl 2-chloro-4-nitrophenyl carbonate. Ethyl 4-chloro-2-nitrophenyl carbonate. Ethyl p-nitrophenyl carbonate. ......... p-Nitroacetanilide ...................... p-Nitrophenyl phosphate. ..............

% % 123 158 114 154 100 106

70 70 39 50 57 50 28 39 15 37

0 0 0 0


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TABLE IV Cleavage of aryl esters by cysleine and glutathione

The cleavage of the nitrophenyl esters was followed spectrophotometrically in a reaction mixture that contained equimolar concentrations of the ester and the sulfhydryl compound in 3 ml of 0.014 M Verona1 buffer, pH 8.1, at room temperature. The hydrolysis of phenyl acetate (44 pmoles) and p-chlorophenyl acetate (24 pmoles) in the presence of cysteine (9.2 pmoles) was measured titrimetrically at pH 8.1. Appropriate blanks were run to correct for spontaneous hydrolysis.

Phenyl ester

4-Chloro-2-nitrophenyl acetate. . 150.0 o-Nitrophenyl acetate.. 73.9 p-Nitrophenyl acetate. 72.0 m-Nitrophenyl acetate.. 31.2 p-Chlorophenyl acetate. 6.25 Phenyl acetate. 1.75 Ethyl 2-chloro-4-nitrophenyl carbonate.. 31.2 Ethyl 4-chloro-2-nitrophenyl carbonate.. 18.1 Ethyl p-nitrophenyl carbonate.. 18.4

-

TABLE V

K, second order

Cysteine Glutathione

liter mow Y-1 min-’

-

131.0 64.3 61.5 35.7

34.3 17.3 17.6

Comparison of rates of cleavage of nitrophenyl esters at pH 8.6 and 8.0

The reaction mixture contained 10 pmoles of cysteine, glutathione, or imidazole, 1 rmole of the aryl ester in 0.1 ml of ethanol, and 40 rmoles of Verona1 buffer, pH 8.0 or 8.6. The total volume was 3 ml. The reactions were followed spectrophotometrically at room temperature. Appropriate blanks were run to correct the spontaneous hydrolysis of the esters. The ratios for the enzyme reactions were calculated from the V,., values.

Ratio of constant at pH 8.6 to constant at pH 8.0

Ester and nucleophil I K (apparent first order)

1.05 o-Nitrophenyl acetate

Imidazole .................. Cysteine .................... Glutathione. ............... Enzyme ....................

m-Nitrophenyl acetate Imidazole .................. Cysteine .................... Glutathione ................ Enzyme ....................

Ethyl p-nitrophenyl carbonate Imidazole .................. Cysteine .................... Glutathione ................ Enzyme. ........................... .I

1.05 1.9 2.45

1.8 2.1

1.05 1.3 2.0

V mar

2.6

2.0

2.6

glutathione have been shown to cleave the ester bond of p-nitrophenyl acetate (5). Since sulfhydryl groups are clearly involved in the enzymatic hydrolysis of p-nitrophenyl acetate (1,2), it was of interest to compare the effectiveness of the sulfhydryl reagents and the dehydrogenase with regard to the esterolysis of various phenyl acetate derivatives. The reactions were followed either

spectrophotometrically or titrimetrically as described in Table IV. When the nitrophenyl acetate derivatives were cleaved by cysteine, about 2 equivalents of acid were produced for every bond cleaved. One equivalent comes from the phenol, and the other, from the decreased basicity of the amine group as the acetyl group is transferred from the sulfur to the nitrogen atom to produce N-acetylcysteine (26). This transformation allowed the cleavage of phenyl acetate and p-chlorophenyl acetate to be followed titrimetrically. With glutathione, approximately 1 equivalent of acid was produced for every phenol group, thereby attesting to the formation of the stable thioester, S-acetylglutathione. If equivalent amounts of the sulfhydryl compound and ester were used, the reaction followed second order kinetics. Un- der first order conditions with a low substrate concentration of 0.07 pmole per ml, at pH 8.0, the enzyme cleaves p-nitrophenyl acetate 700 times more efficiently than cysteine on a molar basis. It should be noted that the enzymatic reaction is an over-all hydrolysis, whereas the reaction with cysteine only involves acylations.

The relative rates of cleavage of the phenyl acetate derivatives by the sulfhydryl compounds are consistent with the order of the pK values of the corresponding phenols as listed in Table I. In general the comparative rates of cleavage by cysteine and glutathione also approximated the relative V,,, values obtained with the enzyme. However, 4-chloro-2nitrophenyl acetate was cleaved more rapidly than p- and o-nitrophenyl acetate by the sulfhydryl compounds, whereas the NAD-free dehydrogenase hydrolyzed these two aryl esters at the same rate. Some differences are also noted in the cleavage of the phenyl carbonate derivatives. These variations indicate that the rates of the enzymatic cleavage are dependent on additional factors, such as substrate specificity and affinity. The cleavage rates with the sulfhydryl compounds (Table IV) correlate better with the enzymatic rates at subsaturation levels of substrate (Table III) than with the comparative V,,, values (Table I). Since the correlation should be with acylation rates rather than hydrolysis, the better correspondence with Table III suggests that the rates in Table III may reflect rates of acylation.

In order to determine what functional group might account for the increase in enzymatic activity with pH, three nitrophenyl esters were cleaved with IO-fold excesses of cysteine, glutathione, and imidazole at pH 8.0 and 8.6. The pseudo-first order rate constants were determined, and the ratios of the rate constants are presented in Table V. The pH does not affect the hydrolysis of the esters by imidazole, since the ratios of the rate constants are consistently 1. With the sulfhydryl compounds, the rate of aryl ester cleavage increased when the pH was raised from 8.0 to 8.6, as indicated by ratios greater than 1. The ratios of the rate constants for glutathione most closely resemble the ratios observed with the dehydrogenase. The results obtained with o- and m-nitrophenyl acetate and ethyl p-nitrophenyl carbonate suggest that the initial cleavage of the aryl ester bond involves a cysteine residue rather than an imidazole residue. Under the conditions described in Table V, S-acetylglutathione exhibited negligible cleavage activity towards o-nitrophenyl acetate.

Efect of Iodmobenzoate and Arsenite on Esbrolytic Reaction- Iodosobenzoate, which is known to oxidize the sulfhydryl groups of enzymes, can stimulate the so-called phosphatase activity of the dehydrogenase and thereby increase the rate of hydrolysis of acetyl phosphate and 1,3-diphosphoglyceric acid (27, 28). Io- dosobenzoate was found to have the opposite effect on the estero-


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E. J. Olson and J. H. Park 2321

Moles of o-iodosobenzoote per Mole Enzyme

FIQ. 2. The inhibition of p-nitrophenyl acetate hydrolysis by o-iodosobenzoate. The NAD-free dehydrogenase, 1.0 mg (0.007 Nmole), was incubated with the inhibitor at a ratio of 1 to 5 moles of o-iodosobenzoate per mole of enzyme for 10 minutes at room temperature in 3 ml of 0.014 M Veronal, pH 7.5. The hydrolytic reaction was started by adding 0.8 pmole of p-nitrophenyl acetate in 0.2 ml of 50% ethanol. Corrections were made for the spontaneous hydrolysis of the substrate under comparable conditions.

lytic activity of the dehydrogenase and to inhibit stoichiometri- tally the hydrolysis of p-nitrophenyl acetate. This inhibition, which was complete in 10 minutes, could not be overcome by raising the substrate level. Under the conditions described in Fig. 2, 3 or 4 moles of iodosobenzoate per mole of enzyme completely blocked the enzymatic activity. When the NAD-free dehydrogenase was treated with 4 moles of iodosobenzoate and then assayed for dehydrogenase activity with 3-phosphoglyceraldehyde, NAD, and arsenate (20), the oxidation reaction was almost completely inhibited.

The oxidation and partial identification of the sulfhydryl groups have been studied with 14C-iodoacetic acid (Table VI). When the native or NAD-free dehydrogenase was incubated with YLiodoacetic acid, 3 of the 12 sulfhydryl groups were selectively labeled (3). In the presence of 3 moles of iodosobeneoate, the labeling with iodoacetic acid was inhibited, indicating that the iodosobenzoate had oxidized the sulfhydryl group at the active site. When the enzyme was incubated with iodoacetic acid in the presence of urea, it was possible to label all 12 sulfhydryl groups. However, after pretreatment with iodosobenzoate, only 6 sulfhydryl groups were then available.’

1 We are grateful for the advice given to us by Dr. J. I. Harris and R. Perham, who are engaged in detailed studies of sulfhydryl oxidations with o-iodosobenzoate.

TABLE VI

Efects of o-iodosobenzoate and arsenite on carboxymethylation of d-phosphoglyceraldehyde dehydrogenase with W-IAA

In the first experiment 0.2 pmole of 3-phosphoglyceraldehyde dehydrogenase was incubated with 0.6 pmole of iodosobenzoate for 10 minutes in 1.5 ml of 0.12 M Tris buffer, pH 8.0. r4C-Iodo- acetic acid (4.0 pmoles, 1.5 X lo8 c.p.m. per pmole) was added to this incubation mixture and to a control enzyme solution which had not been treated with iodosobenzoate. After 1 hour at O”, the enzyme was precipitated with 7.0 ml of a mixture of cold acetone and 1 N HCI (20:1), washed five times with this mixture, twice with acetone, and once with ether, and then dried in air. Suitable weighed samples were digested with pepsin, and the radioactivity bound to the protein as 14C-carboxymethyl groups was counted in a liquid scintillation counter. In order to main- tain a continuity with previous experiments of this type (3), the molecular weight of the enzyme was taken to be 126,000.

In the second experiment, the enzyme was treated with iodosobenzoate and IAA as described above. Solid urea (1.0 g) was then added to the solution, which was kept at room temperature for 2 hours. The enzyme was precipitated and washed, and the number of I%?-carboxymethyl groups on the protein was then determined.

The conditions for the third experiment were the same as those in the first experiment. The enzyme was preincubated with the designated concentrations of arsenite for 10 minutes at room temperature before the addition of “C-IAA.

Experi- ment

I_ Additions

o-1odo.w benzoate

n&s/m& enzyme

0

3

0 3

Arsenite “C-IA‘4

M moles/mole cnzytnc

20 20

20

.I-

20

0 20 1 x 10-a 20 1 x 10-1 20

Urea

0

+

0

L

Ratio of “C-CdKlXy- methyl groups

to enzyme

2.5:1 0:l

11.5:1 6.1:l

2.6:1 2.5:1 2.5:1

Arsenite at a concentration of 1 X lo-’ M has been shown to inhibit enzymes such as aldehyde dehydrogenase (29) and cy-keto acid oxidase systems (30), which are thought to involve vicinal dithiol groups in the catalytic processes. Since the active site of the dehydrogenase can be oxidized with iodosobenzoate and contains two cysteine moieties, the hydrolytic activity of the dehydrogenase was tested in the presence of varying concentrs- tions of arsenite. No inhibition of the esterolytic reaction could be observed at concentrations up to 1 X lo+ M arsenite. More- over, arsenite does not interfere with the carboxymethylation of the enzyme with *4C-IAA2 (Table VI). Although the hydrolysis of p-nitrophenyl acetate is not effected by arsenite, concentrations of 1 X lo-* M arsenate produces a 45% inhibition of this esterolysis (1). These data are consistent with the results of Jakoby (29), who was unable to inhibit the oxidation activity of glyceraldehyde phosphate dehydrogenase with arsenite.

Efect of pH on Acetyl-enzyme Formation-To investigate the possibility that a histidine moiety was present at the esterolytic

* The abbreviations used are: IAA, iodoacetic acid; DFP, di- isopropyl fluorophosphate.


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2322 Esterolytic Activity of S-Phosphoglyceraldehyde Dehydrogenase Vol. 239, No. 7

site of the enzyme, the hydrolysis of nitrophenyl acetate derivatives was studied in the pH range which encompasses the pK of the imidazole group of histidine. It was previously found that near neutrality at 0”, the enzymatic hydrolysis of p-nitraphenyl acetate proceeds with an initial rapid liberation of p-nitrophenol followed by a slow, linear rate of hydrolysis (1). When p-nitrophenol liberation was plotted against time, the extrapolation from the terminal phase of hydrolysis to zero time gave inter- cepts which ranged from 2.7 to 3.5 moles of p-nitrophenol per mole of enzyme. Furthermore, when incubated with Y-labeled p-nitrophenyl acetate under these conditions, the enzyme can be acetylated with approximately three acetyl groups per mole of enzyme. Therefore, at 0” near neutrality, the enzymatic hydrolysis of p-nitrophenyl acetate probably proceeds with an initial rapid acetylation of the enzyme followed by a slower, rate-limiting deacylation reaction.

In Fig. 3 the enzymatic liberation of p-nitrophenol from p-nitrophenyl acetate was plotted against time at pH 6.4 and 7.6. From the comparison of the curves at two different substrate levels, it is apparent that the course of the esterolytic reaction was more dependent on the substrate level at pH 6.4 than at pH 7.6. The data suggest that the aflinity of the enzyme for p-nitrophenyl acetate was reduced as the pH was lowered from 7.6 to 6.4. In these experiments the slightly low acetyl to enzyme ratio of 2.4: 1 is possibly related to the malonate buffer.

.lO-

.09-

.08-

0 4.4pmoles substrate,pH 7.6

@ 4.4pmoles substrate, pH 6.4

v 1.1 Nmolds substrate,pH 7.6

A 1.1 pmales substrote,pH 6.4

, I

4 8 12 16 20 24

Minutes

FIG. 3. Effect of pH and substrate concentration on the hydrolysis of p-nitrophenyl acetate. The reaction mixture contained 30 pmoles of Tris and 30 pmoles of malonate, buffered at either pH 6.4 or 7.6, and 2.3 mg (0.016 ,umole) of the NAD-free dehydrogenase. The reaction was started by adding either 4.4 or 1.1 pmoles of p-nitrophenyl acetate in 0.2 ml of 50% ethanol. The liberation of phenol was followed in a jacketed Beckman DU spectrophotometer at 400 rnp. The temperature was kept at O-4” throughout the course of the experiment. Appropriate blanks were used to correct for spontaneous hydrolysis.

1.0

1

0.8

r 0.2 1

Q

+ 4-chloro,2-nitrophenyl acetate, I.lpmOleS

+ 2-chloro,4-nitrophenyl acetate, I.IpmOleS

-o-p- nitrophenyl acetate, I, I )Imoles

: 6:3 6.8 7.1

PH

7.6

FIQ. 4. The effect of pH on the acylation of the enzyme. The apparent first order rate constants for the acylation of the enzyme were determined at 4’ in 0.01 M Tris-0.01 M malonate buffer with 1.1 or 4.4 &moles of substrate and 3 mg (0.021 pmole) of NAD-free dehydrogenase in a total volume of 3.0 ml. The concentration of the substrates in the reaction mixture was determined by solubility factors. The calculations were based on the premise that three esterolytic sites were available per mole of enzyme throughout the entire pH range. The initial velocities of the first minute were used to determine the rate constants.

The curves obtained with higher substrate level show that the deacylation is also facilitated by raising the pH.

Similar experiments were conducted with 2-chloro-4-nitrophenyl acetate and 4-chloro-2-nitrophenyl acetate as substrates. First order rate constants were calculated from readings taken during the first minute of the time course reaction (Fig. 4). As the pH was lowered, a marked decline in the rates of enzyme acylation could be observed with 2-chloro-4-nitrophenyl acetate, 4-chloro-2-nitrophenyl acetate, and p-nitrophenyl acetate. On the basis of the results presented in Figs. 3 and 4, it is considered that the formation of an imidazolium ion may reduce the aflinity of the enzyme for nitrophenyl esters. It is apparent that the chloronitrophenyl acetate derivatives were more effective acylat- ing agents than p-nitrophenyl acetate. This difference was also demonstrable at pH 6.4 and 6.8, where both 2-chloro-4-nitrophenyl acetate and 4-chloro-2-nitrophenyl acetate gave acetyl to enzyme ratios which were higher than those obtained with p-nitrophenyl acetate. The interpretation of these results is considered below.

DISCUSSION

Mechanism of Acylation of S-Phosphoglyceraldehyde Dehy- drogenase-The isolation and ammo acid sequence studies of the active site of 3-phosphoglyceraldehyde dehydrogenase firmly established the formation of an S-acetyl bond in the catalytic


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mechanism for the hydrolysis of p-nitrophenyl acetate (3). In its simplest form the reaction can be written as follows.

3 p-Nitrophenyl acetate + enzyme + (S-acetyl),-enzyme +

1 3 p-nitrop’henol

J&O 3 acetic acid + enzyme

The available data are consistent with the idea that the liberation of the phenol is brought about by a nucleophilic attack of the sulfhydryl group on the carbonyl carbon of the acetate moiety. Various thiols, such as mercaptoethanol (31), o-mercaptobenzoic acid (32), cysteine, and glutathione (5), act as nucleophilic reagents in the cleavage of p-nitrophenyl acetate. Thus it is highly probable that the sulfhydryl groups of the three active sites of the dehydrogenase carry out a similar type of cleavage. The enzyme is 700 times more effective than cysteine or glutathione in cleaving p-nitrophenyl acetate. Three sulfhydryl groups on the native enzyme can be specifically acetylated with p-nitrophenyl acetate and are highly reactive as compared to the nine other sulfhydryl groups (3).

A nucleophilic attack by a sulfhydryl group on the various phenyl acetate derivatives should be favored by the substitution of electron-withdrawing groups in the phenyl ring. An increased electron-withdrawing capacity of the phenyl moiety enhances the electropositive nature of the carbonyl carbon and makes it more susceptible to hydrolysis. The data of Tables III and IV clearly show that the rate of cleavage of nine aryl esters by cysteine and glutathione is related to the pK, the rate of enzymatic hydrolysis at subsaturation levels of substrate, and the rate of spontaneous hydrolysis of the esters. The maximal rates of enzymatic hydrolysis of these derivatives also increase as the pK of the corresponding phenols decrease (Table I). Moreover, p-nitrophenyl acetate is hydrolyzed more rapidly than ethyl p-nitrophenyl carbonate, in which the resonance or the carbonyl group is altered (Table II). Small differences between the relative rates of hydrolysis with the sulfhydryl compounds or the enzyme could be accounted for on the basis of enzymatic specificity. For example, phenyl acetate, which is not hydrolyzed by the enzyme, is slowly cleaved by cysteine. It is interesting to note that p-nitroacetanilide and p-nitrophenyl phosphate are not cleaved by cysteine, glutathione, or the dehydrogenase.

A similar line of reasoning was employed by Bender and Naka- mura (33) in demonstrating that the acylation of the serine moiety of a-chymotrypsin resembles a nucleophilic reaction. The variation of the rate constants for the enzymatic acylation with substituted phenylacetate derivatives such as p-nitro, p-aldehydo, p-acetyl, m-nitro, m-aldehydo, m-phenyl, and o-nitrophenyl acetate paralleled the effect of structure on the reactivity of the phenyl acetates in a number of nucleophilic reactions. It was clear that electron-withdrawing substituents generally facilitated the acylation of a-chymotrypsin.

Numerous investigations involving the over-all enzymatic hydrolyses of phenyl acetate derivatives have qualitatively indicated that the effects of substituents in the phenyl ring run parallel in enzymatic and nonenzymatic nucleophilic reactions (34). Typical investigations include studies on the hydrolysis of substituted phenyl acetates catalyzed by wheat germ lipase (7), the hydrolysis of various sulfates by an aryl sulfatase (34), and cleavage of phenyl /?-n-glucosides by emulsin (35). It should be noted that certain exceptions to this generalization have

been observed in experiments with phenyl acetate derivatives and various cholinesterases (36, 37).

Since the V,,, for hydrolysis of the phenylacetate derivatives catalyzed by 3-phosphoglyceraldehyde dehydrogenase is generally related to the pK or lability of the aryl ester bond (Tables I and III), it appears that for most esters the rate-limiting step in the over-all esterolysis at pH 8.6 is the cleavage of the aryl ester bond and not the deacylation of the enzyme. The exceptions to this relationship are 2-chloro-4nitrophenyl acetate and 4-chloro- 2-nitrophenyl acetate. They may not be hydrolyzed more rapidly than o- and p-nitrophenyl acetate at pH 8.6, because the deacylation of the enzyme is rate-limiting when the system is saturated with these substrates. The possibility of product inhibition has been essentially ruled out since 2-chloro-4-nitrophenol does not inhibit the hydrolysis of 2-chloro-4-nitrophenyl acetate.

There are conditions under which significant differences can be observed between the nitrochlorophenyl esters and the nitrophenyl esters: (a) at pH 7.5, where the rates of hydrolysis are relatively low, 2-chloro-4-nitrophenyl acetate is hydrolyzed over 1.5 times as rapidly as p-nitrophenyl acetate (Fig. 1); (5) when the enzyme is not saturated with substrate, as in Table III, the disubstituted phenyl acetate derivatives are hydrolyzed more rapidly than the monosubstituted esters; (c) the acylation of the enzyme at pH 6.3 to 7.5 proceeds more rapidly with the chloronitro derivatives than with p-nitrophenyl acetate (Fig. 3). Under these circumstances the disubstitutions with electrophilic moieties favor cleavage. This is perhaps most clearly illustrated by the fact that ethyl 2-chloro-4-nitrophenyl carbonate is hydrolyzed much more rapidly than ethyl p-nitrophenyl carbonate (Table II).

A large body of evidence has shown serine to be involved in the hydrolyses catalyzed by proteolytic enzymes. Since a serine moiety is adjacent to the active sulfhydryl group in the catalytic site of the dehydrogenase, it was thought that the inhibitors such as iodoacetic acid, iodoacetamide, and p-chloromercuribenzoate might combine with the sulfhydryl group at the active site and bring about the inhibition of esterolysis by a steric hindrance of the neighboring serine group. In order to rule out this possibility, another type of sulfhydryl reagent was tested, namely, iodobenzoate, which is considered to oxidize the sulfhydryl groups to disulfide bridges. Iodosobenzoate was found to inhibit the esterolysis stoichiometrically (Fig. 2). The experiments of Table VI showed that 3 moles of iodosobenzoate per mole of enzyme oxidized 6 of the 12 sulfhydryl groups of the enzyme.

Since the amino acid sequence of the active site of the dehydrogenase has been shown to contain two cysteine moieties (3) and the sulfhydryl groups can be oxidized by iodosobenzoate, it was thought that arsenite might inhibit the esterolytic reaction. However, arsenite showed no effect even at a concentration of 1 x 10m2 M, which is 100 times higher than that required for the inhibition of enzymes with vicinal dithiols (30). Neither the dehydrogenase activity of the enzyme (29) nor the carboxymethylation with 14C-IAA (Table VI) is inhibited by arsenite. Thus a correct positioning of the two -SH groups in the active site appears to be essential for oxidation or binding of various reagents.

The correlation between the inhibition of the esterolysis and the dehydrogenase activity of the enzyme by iodosobenzoate and other sulfhydryl inhibitors (1) indicates that at least a part of the


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2324 Esterolytic Activity of S-Phosphoglyceraldehyde Dehydrogenase Vol. 239, No. 7

pH 5.0 l I) pH7.OCICo) or pH 7.0 (ICb) pH 8.3 (III)

6 b NO,330-kc”,

2 f: b;” < ~cY;*gzsH a(+./gcH3

FIG. 5. Upper, diagrammatic representation of the enzyme. Lower, suggested mechanism for hydrolysis of p-nitrophenyl acetate

site for oxidation activity is involved in the esterolytic site. In addition, 3-phosphoglyceraldehyde has been shown to inhibit the over-all hydrolysis of p-nitrophenyl acetate, the acetyl enzyme formation (l), and the carboxymethylation of the active site with 14C-IAA (3).

The iodosobenzoate inhibition of the esterolytic reaction is at variance with the stimulation of the acetyl phosphatase activity of the dehydrogenase by this reagent (27). Moreover, acetyl phosphatase activity is not inhibited by IAA (8). These two facts suggest that esterolytic activity involves a thioester intermediate, while acyl phosphatase activity does not.

Preliminary results indicate that histidine may be involved in the acylation of the enzyme with phenyl acetate derivatives. The increase in the affinity of the nitrophenyl esters for the enzyme as the pH is raised from 6.3 to 7.6 could be due to the removal of the imidazolium ion and interaction of the imidazole ring with the active sulfhydryl group. Since the imidazole group may also be involved in the deacylation reaction, the role of histidine is considered in more detail in the next section.

Deacylation of S-Phosphoglyceraldehyde Dehydrogenase-The over-all mechanism for enzymatic esterolysis involves two steps, acylation and deacylation, which are equally important in ac- counting for the greater efficiency of the hydrolytic enzymes as compared to model systems. For both chymotrypsin and 3-phosphoglyceraldehyde dehydrogenase, the mechanism of the acylation step has been rather well formulated, the amino acid sequence of the site for acylation has been determined, and the results have been substantiated by inhibitor studies with DFP or IAA (3, 38). However, the mechanisms for deacylation are in a more preliminary state. In the case of chymotrypsin a great body of evidence has strongly implicated histidine as the primary factor in deacylation. In a recent review article Jencks (39) has considered this proposal with caution: “Although there is much evidence for the involvement of imidazole groups in the active sites of enzymes, no evidence has yet been obtained, un- fortunately, which demonstrates that imidazole is a nucleophilic catalyst in any enzymic reaction.” Statements corroborating this opinion also appear in Kosower’s book, Mobcular Biochemis- try (40). The factors involved in the deacylation step with the

dehydrogenase are even less thoroughly investigated or understood. Bender (41) concluded from his studies with ficin, papain, and model thiol compounds that any mechanism for the catalysis of ester hydrolysis by sulfhydryl enzymes is extremely complicated and must involve a number of factors.

We are, therefore, forced to consider the possibilities for removal of the acetyl group from the surface of the dehydrogenase. The most reasonable candidates are serine and histidine. Serine may be involved, since both the esterase and dehydrogenase activities are inhibited by DFP. However, it should be noted that the required concentrations of 1.4 X 10m4 and 5.6 x low4 M

are extremely high (1). In view of the effects of contaminants in various DFP samples, these results should be viewed with reservation (42). Several lines of evidence have implicated a histidine moiety in the catalytic mechanism. First, the rates of acylation and deacylation of the enzyme increase between pH 6.3 and 7.6 (Fig. 3). Second, the acetyl-enzyme formed with acetyl phosphate and an enzyme with fully reduced sulfhydryl groups has a spectrum that more closely resembles N-acetylimidazole with a maximum around 240 rnp than a thioester (43). Third, photooxidation of the enzyme inhibits the esterase and dehydrogenase activities.3 These findings are subject to the usual limitations of interpreting pK measurements, spectral observations, the nonspecificity of photooxidation, and inhibition produced by distortion of the enzyme with inactivating agents. Nonetheless, a mechanism for esterolysis involving sulfhydryl group and histidine which is consistent with these and other characteristics of the reaction can be designed.

On the first line of Fig. 5, the enzyme is diagrammatically represented at three different pH values. At pH 5.0 (I), the sulfhy- dry1 group is not charged, but the histidine exists as the imidazolium ion. At pH 7.0 (ZZa and ZZb), the formation of a hydrogen bond between the sulfhydryl group and the uncharged imidazole ring is now feasible and might be facilitated by steric factors in the conformation of the protein. The polarization of this structure would cause the sulfur to be the nucleopbilic agent which attacks the positively charged carbonyl carbon of p-nitrophenyl

8 P. Elodi, personal communication.


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HPO;

z Cys-S - C-CHS

::f: + -0-POCCH3

I o-

f3 t HO- CCHJ t HAsO;

FIG. 6. Suggested mechanism for the dehydrogenase reaction with acetaldehyde as the model substrate in the place of 3.phosphoglyceraldehyde.

acetate. At pH 8.3 (III), the CyS- would be an even more effec- pyriclmium moiety of the NAD+. The NAD+ may be useful tive nucleophilic agent. The advantage of the relatively stable because of its charge and its effects in maintaining the proper thioester intermediate can be understood in terms of the subse- three-dimensional configuration of the enzyme (53). quent transfer and hydrolysis step, which would be accelerated by This mechanism is rendered more plausible by consideration of a certain degree of steric restriction required for very rapid intra- the model reactions for imidazole designed by the organic chem- molecular catalysis (44, 45). The suggested imidazole group is ists. (a) The thioester linkage is extremely sensitive to imid- a particularly good nucleophil for hydrolysis of thioesters (46). azole. Ethyl thioacetate but not ethyl acetate can be cleaved Moreover, there is a chemical precedent for this type of catalysis by imidazole (44). (b) An imidazole-dependent transfer of the because imidazole catalyzes the removal of a proton from the acyl group of acetyl phosphate to a sulfhydryl compound to form attacking thiol in reactions with the acyl group of acetylimidaz- an acetyl thioester has been described by Jencks and Carriuolo ole (47). In accordance with this scheme, increasing the pH (54). Moreover, arsentate, phosphate, and NHzOH can accept from 6.3 to 8.9 would also promote the deacylation of the enzyme the acetyl group from the acetylimidazole intermediate. (c) An brought about by hydroxyl ions. This proposed mechanism is ingenious model for intramolecular hydrolysis by a nucleophilic somewhat analogous to that involving the serine-imidazole inter- imidazole group and a p-nitrophenyl ester has been constructed action of the active site of chymotrypsin (48, 49). by Bruice and Sturtevant (44), namely, p-nitrophenyl-y-(imid-

Since many of the characteristics of the esterase activity are azolyl)butyrate. paralleled in the classical dehydrogenase reaction, this diagrammatic mechanism must coincide with the facts concerning the oxidative phosphorylation. An attempt has been made to include these concepts in a mechanism for dehydrogenation (Fig. 6). This scheme accounts for the general characteristics of the over-all reaction. The esterolysis and oxidation occur at the same site, as indicated by the inhibition of the acylation of the enzyme by 3-phosphoglyceraldehyde and glyceraldehyde (1). N-N After the hydrogen transfer, the NADH is less firmly bound than =-O the NAD+ (50), and is therefore replaced by NAD+, which is

H,O ’

known to facilitate transfer reactions (51, 52). The imidazole

v -o*-

group can now function as a nucleophilic reagent and transfer the acyl group from the N-acetylhistidine position to a number of The steric restraints imposed by thii intramolecular model m-

acceptors such as phosphate, arsenate, or the sulfhydryl com- crease the hydrolytic rates to equal that of chymotrypsin. In- pounds, glutathione and CoA (51). A simple reversal of this termolecular imidazole catalysis of p-nitrophenyl acetate hy-

process could account for the “transferase activity” of the en- drolysis is 100 to 1000 times slower than chymotrypsin. An zyme. The sulfur group would act as a stabilizer for the acyl equivalent steric restraint on the enzyme surface could account moiety during the exchange of acceptors. The anions, phos- for the rapidity of enzymatic catalyses. Bruice has also investi- phate and arsenate, may be more effective acceptors than the gated the intramolecular nucleophilic catalysis of the acyl thiol, sulfhydryl compounds by virtue of the positive charge on the n-propyl-y-(4-imidazolyl)thiobutyrate.


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This acyl thiol is hydrolyzed lo6 to 10’ times as fast as a normal thioester would react under the same conditions with hydroxide ions (45). Thus, if an acyl thiol were formed on the enzyme so that the steric relationship of the ester bond to the histidine moiety were analogous to that in the model, the deacylation or transfer reactions could occur at the enzymatic rates.

It is tempting to extend this mechanism to cover the acyl phosphatase activity of the enzyme. If the sulfhydryl group were removed by oxidation with iodosobenzoate, the histidine portion of the site might bring about the hydrolysis of acetyl phosphate. However, the rate of hydrolysis increases as the pH is lowered from 8.0 to 6.0 (27). Another theory may have to be proposed for this aberrant activity.

The mechanism as written in this paper is far from complete. The factors involved in NAD binding, NADHX formation (55), and diaphorase activity (56) are not even considered in this hy- pothesis. It is only hoped that some of the present notions may be pertinent to the final solution of the problems posed by this versatile enzyme.

SUMMARY

1. 3-Phosphoglyceraldehyde dehydrogenase, which is free of enzyme-bound nicotinamide adenine dinucleotide catalyzes the hydrolysis of p-nitrophenyl acetate, o-nitrophenyl acetate, m-nitrophenyl acetate, p-chlorophenyl acetate, o-chlorophenyl acetate, 2-chloro-4-nitrophenyl acetate, 4-chloro-2-nitrophenyl acetate, ethyl p-nitrophenyl carbonate, ethyl 2-chloro-4-nitrophenyl carbonate, and ethyl 4-chloro-2nitrophenyl carbonate.

2. This esterolytic activity is restricted to aryl esters. The rate of the reaction is related to the electron-withdrawing capacity of the phenolic moiety.

3. The esterolytic and dehydrogenase functions of the enzyme are inhibited by treatment of the protein with iodosobenzoate. This suggests that the same sulfhydryl groups are required for both catalytic activities. By contrast, the acetyl phosphatase activity of the enzyme is enhanced by treatment with iodosobenzoate.

4. The cleavage of aryl esters by the dehydrogenase, cysteine, and glutathione was studied. The enzymatic reaction appears to involve a nucleophilic attack of the sulfhydryl group of a cysteine residue on the carbonyl carbon of the acetate moiety of the ester.

5. Preliminary studies indicate that a histidine moiety may also be involved in the enzymatic hydrolysis of the aryl esters.

6. A mechanism is suggested in which interactions between cysteine and histidine could in part account for the esterolytic, oxidative, and transfer activities of the dehydrogenase.

Acknowledgments-The authors would like to thank Dr. Sid- ney Colowick for helpful discussions throughout the course of

5. DIRKS, B. M., AND BOYER, P. D., Cereal Chem., 28,483 (1951). 6. HARTELY, B. S., AND KILBY, B. A., Biochem. J., 50,672 (1952). 7. GAWRON, O., GRELECKI, C. J., AND DUGGAN, M., Arch. Bio-

them. Biophys., 44, 455 (1953). 8. PARK, J. H., AND KOSHLAND, D. E., J. Biol. Chem., 233, 986

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Erik J. Olson and Jane Harting Park3-Phosphoglyceraldehyde Dehydrogenase

Studies on the Mechanism and Active Site for the Esterolytic Activity of

1964, 239:2316-2327.J. Biol. Chem.

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