THE JOURNAL OF B~LOQICAL CHEMISTRY Vo1.245, No. 11,Issue of June 10, pp. 2946-2953, 1970

Printed in U.S.A.

Enzymatic Characterization of the N-Acetylation of

3-Phosphoglyceraldehyde Dehydrogenase

by Acetyl Phosphate*

(Received for publication, February 2, 1970)

JANEHARTINGPARK,DENISC. SHAWJ ELIZABETH MATHEW,ANDBLANCHE P. MERIWETHER

From the Department of Physiology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37203

SUMMARY

Acetyl phosphate or p-nitrophenyl acetate acetylates a specific cysteine residue in the active center of d-phospho- glyceraldehyde dehydrogenase crystallized from rabbit muscle. This reaction occurs more easily with the enzyme from rabbit than from yeast. At pH 7.0 and 0” the cysteine residue is readily acetylated and forms a common intermedi- ate in the dehydrogenase, transferase, and esterase reactions. On warming or raising the pH to 8.5, the acetyl groups migrate from the cysteine to a specific lysine moiety by a S-N transfer reaction. Three to 4 cysteine or lysine residues can be acetylated per molecule of rabbit muscle dehydrogenase (mol wt 140,000). The distribution of the acetyl group be- tween these 2 residues is affected by the pH, substrate con- centration, and time of incubation. A more specifically and completely labeled iV-acetyl enzyme can be prepared with acetyl phosphate than with p-nitrophenyl acetate.

N-Acetylation of the enzyme impairs DPN binding and, depending on the assay conditions, produces varying degrees of inhibition of the dehydrogenase activity. On the other hand, DPN inhibits the N-acetylation of the enzyme. When DPN is added to the N-acetylated dehydrogenase, the co- enzyme protects against progressive inactivation of the pro- tein with time.

3-Phosphoglyceraldehyde dehydrogenase has two unusual properties as an enzyme. First, by a variety of experimental manipulations the dehydrogenase can be converted into a trans- acylase, phosphatase, or an esterase with acetyl phosphate and p-nitrophenyl acetate as substrates (l-5). The physical state of the enzyme is critical, and the various activities are often mutually exclusive (4). Second, acetyl phosphate or p-nitro- phenyl acetate can acetylate a specific cysteine residue at the active site, and under certain conditions the acetyl group can

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

$ On leave of absence from the Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, Austraiia.

then be transferred to a specific lysine moiety or to another cysteine residue (6-10). The sequences for the purified peptides containing the S-acetyl cysteine at the active site, the N-acetyl lysine residue, and the alternate cysteine residue have been de- termined (6, 8-10). This is an interesting demonstration of a controlled migration of the substrate from the active site to other residues on the enzyme surface.

In this paper, the characteristics of the N-acetylation reaction of the lysine residue by acetyl phosphate and p-nitrophenyl acetate are described in detail. p-Nitrophenyl acetate is a con- venient substrate for kinetic analyses because its hydrolysis can be readily assayed spectrophotometrically. However, a more completely labeled N-acetyl dehydrogenase can be prepared with acetyl phosphate as substrate rather than p-nitrophenyl acetate. In general, acetyl phosphate is a more useful substrate for the study of the biological reaction of oxidation and phosphorylation catalyzed by the enzyme because it is more closely related than p-nitrophenyl acetate to the natural substrate, 1, S-diphospho- glyceric acid. For these reasons, the properties of the acetyla- tion reactions with acetyl phosphate are detailed in this paper.

In earlier studies, Mathew and Park found that the coenzyme, DPN, inhibits the N-acetylation of the enzyme, and, conversely, N-acetylation interferes with coenzyme binding (7, 11). In studies on the crystalline enzyme from pig muscle, Polgar con- firmed the inhibition of the N-acetylation by the coenzyme, but he did not find any impairment of DPN binding with an N- acetylated enzyme (12). In an attempt to clarify this discrep- ancy, we have repeated the DPN binding studies with the N- acetylated enzyme and have also investigated in greater detail the dehydrogenase activity of the N-acetylated enzyme.

MATERIALS AND METHODS

Enzymes and Proteins-3-Phosphoglyceraldehyde dehydro- genase from rabbit muscle was recrystallized twice in the presence of 0.001 M EDTA (13). Enzyme-bound DPN was removed with activated charcoal (14), and the ratio of the optical density read- ings at 280 and 260 rnp varied from 1.75 to 1.9. For the cal- culations in this paper, the value used for the molecular weight was 140,000 (15). The preparations of 3-phosphoglyceraldehyde dehydrogenase from yeast were recrystallized three times in the presence of 0.005 M EDTA and were a gift from Dr. Robert Flora (Vanderbilt University). Crystalline hexokmase from baker’s yeast was kindly supplied by Dr. Irene Schulze (Van-

2946

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Issue of June 10, 1970 J. H. Park, D. C. Xhaw, E. Mathew, and B. P. Meriwether 2947

TABLE I

Comparison of acetylation of different proteins with acetyl phosphate

The crystalline 3-phosphoglyceraldehyde dehydrogenase from

rabbit muscle was treated with charcoal to remove the bound DPN (Azso:Azso ratio, 1.9). The 3-phosphoglyceraldehyde dehy- drogenase from yeast crystallized without enzyme bound DPN

(Azso:Apao ratio, 1.8). The dehydrogenases, aldolase, hexokinase, and albumin were dialyzed for 4 hours against several changes of 0.005 M Tris-0.001 M EDTA buffer, pH 7.0. The proteins in the

designated quantities were incubated with 10 pmoles of W-acetyl phosphate (219,000 cpm per pmole) in 0.1 M Tris at pH 7.0 or 8.5. Total volume, l.Oml. After 30 min at room temperature, the pro-

teins were precipitated with 7 ml of a mixture of acetone-ether-l N HCl, 20:5:1, and kept at -10” for 15 min. The precipitate was washed four timeswith this solution and twicewith 3 ml of acetone

and then dried. Previous experiments had shown that after four washes free acetyl phosphate had been removed. The weighed precipitate was digestedwith 5% pepsin in 0.02 N HCl for 24 hours,

and a suitable aliquot was cormted by the liquid scintillation technique (4).

Protein

%-Phosphoglyceral- dehyde dehy-

drogenase Rabbit muscle Yeast

Aldolase

Hexokinase Albumin

Amount

m# pmolc

14.0 0.1 8010 6690 3.6 3.0

12.0 0.1 3080 2760 1.4 1.2

14.5 0.1 210 388 0.1 0.2

5.0 0.03 99 0.04

14.7 0.24 216 395 0.05 0.1

-

ladioactivity of precipitate

I’C-Acetyl groups bound

,er mole enzyme

a: % molts

derbilt University). Aldolase was obtained from Sigma and bovine serum albumin from Armour.

Substrates and Coenzymes-14C-Acetyl phosphate was prepared by the method of Kornberg (16) with a purity of 60 to 70% as determined by the hydroxamic acid procedure (17), total phos- phate (18), and inorganic phosphate determinations (19). The specific activities were between 200,000 and 400,000 cpm per pmole. 14C-p-Nitrophenyl acetate was prepared with specific activities ranging between 200,000 and 700,000 cpm per pmole (20).

3-Phosphoglyceraldehyde and DPN were products of Sigma. Analytical Methods-The methods for determining bound

i*C-acetyl groups and the amino acid sequence of the radioactive peptides produced by pepsin digestion of the 14C-acetylated en- zymes have been reported previously (4, 6, 7).

RESULTS

Comparison of Acetylation of Different Proteins with Acetyl Phosphate-The specificity of the acetylation reaction of 3-phos- phoglyceraldehyde dehydrogenase with the substrate, acetyl phosphate, is shown in Table I. When the crystalline dehydro- genase from rabbit muscle was incubated with 0.01 M 14C-acetyl phosphate at pH 7.0 or 8.5, the enzyme bound 3 to 4 moles of I%-acetyl groups per mole of enzyme. Most of the preparations bound between 3.0 and 3.5 moles of acetyl groups per mole of enzyme, and only a few bound 3.8 to 4.0 moles. These values

TABLE II

Effect of substrate concentration on acetylation of

%phosphoglyceraldehyde dehydrogenase at pH 4.6, 7.0, and 8.5

The experimental conditions were similar to those outlined in Table I. The dialyzed, DPN-free dehydrogenase (0.1 rmole)

from rabbit muscle was incubated with the designated amount of W-acetyl phosphate for 30 min at room temperature. The pH was maintained at 4.6 with 0.2 M acetate buffer and at pH 7.0 and 8.5 with 0.1 M Tris in a total volume of 1.2 ml. The acetylation reaction was assayed as described in Table I.

“C-A&y1 groups bound per mole DPN-free enzyme “C-A&y1 phosphate

pH 4.6 pH 7.0 pH 8.5

pmolcs ?nolcs

2 1.8 4 1.5 2.8 2.9

10 2.0 3.5 3.2 20 2.1 4.0 3.7

are the same as the number of active sites acetylated by p-nitro- phenyl acetate at pH 7.0 and 0” (4). When the crystalline yeast dehydrogenase was acetylated with acetyl phosphate, it bound only about 1.2 moles of 14C-acetyl groups at pH 7.0 or 8.5 (Table I). With p-nitrophenyl acetate as substrate, these yeast prep- arations bound between 2.2 and 2.9 moles of acetyl groups, when assayed by the spectrophotometric procedure of Taylor, Meri- wether, and Park (5) and Hartley and Kilby (21). The acetyla- tion of the yeast enzyme with p-nitrophenyl acetate (5) or acetyl phosphate is approximately 3 times more difficult than the acetyl- ation of the muscle enzyme. The other two enzymes, aldolase and hexokinase, and albumin showed relatively low levels of labeling.

E$ect of Substrate Concentration and pH on Acetylation of S- Phosphoglyceraldehyde Dehydrogenase with Acetyl Phosphate- Table II summarizes the results of labeling the dehydrogenase with various concentrations of acetyl phosphate at different hydrogen ion concentrations. As the substrate concentration was increased, the number of acetyl groups bound per molecule of enzyme approached the maximal value of about 4.0 at pH 7.0 and 8.5 but not at pH 4.6.

,4 more informative picture of the acetylation reactions at the different pH values was obtained when the acetyl enzyme com- plexes were examined by radioautography (Fig. 1). Enzyme acetylated with acetyl phosphate (0.01 M) at pH 4.6, 7.0, and 8.5 was digested with pepsin, and the IGpeptide patterns were com- pared by electrophoresis. The peptides obtained from the acetyl enzyme prepared at pH 4.6 (Panel 1, Bands Sl to S7) cor- responded to fragments of the active center octadecapeptide (6) containing 14C-acetyl groups in thioester linkage with cysteine residue 8.

“yOCHB

Lys-Ile-V$l-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-Cy~-Le~~-Ala-Pro- 1 8 12

Leu-Ala-Lys 18

In the case of the pH 7.0 peptides, additional bands, Nl, N2, and N3 appeared corresponding to peptide fragments in which the acetyl Froup was attached to the e-amino group of lysine (7).

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N-Acetylation of 3-Phosphoglyceraldehyde Dehydrogenase Vol. 245, No. 11

FIG. 1. Radioautograph of I%-acet,yl peptides from 3-phospho- glyceraldehyde dehydrogenase acetylated with KLacetyl phos- phate (-4cP) at pH 4.5, 7.0, and 8.5. The DPN-free enzyme (0.1 pmole) was incubated with I%!-acetyl phosphate (10 rmoles) for 45 min at room temperattIre. The number of 14C-acetyl groups bound per mole of enzyme was 2.9 at pH 4.6,3.6 at pH 7.0, and 3.43 at pH 8.5. The pepsin digestions were carried out as described in Table I. After lyophilization, 2.0mg of the sample were dissolved in 40 pl of water, streaked on a 4-cm origin on Whatman No. 1 paper, and then ionophoresed in pyridine-acetate buffer, pH 3.5, for 1 hour at 3000 volts. The paper was placed in direct contact with a Blue Brand x-ray film and left in a press until the appropri- ate time for developing.

With the pH 8.5 peptides, the N-acetylated fragments pre- dominated (Panel 3). The amino acid sequences of N-acetylated peptides, as previously determined (7, S), are shown below:

Peptide :

NH14COCH3

Nl: Lys-Thr-Val-Asp-Gly-Pro-Ser-Gly-Lys-I,eu 4 12

NH”COCH 3

N2: Ala-Thr-Gln-Lys-Thr-Val-Asp-Gly-Pro-Ser-Gly-~~~-Leu 4 12

NH”COCH,

N3 : Ala-Thr-Gln-Lui 4

E$ect of Temperature, DPN, and Substrate Concentration on

Acetyl Enzyme Formation--Since the acetylation of the dehydro- genase with p-nitrophenyl acetate is usually performed at 0” (4, 6), the acetylation reaction with acetyl phosphate was also ex- amined at this temperature. With the DPN-free enzyme and the lowest amount of acetyl phosphate (4 pmoles), the acetyla- tion reaction was substantially faster at 25” than at 0”. At the highest level of acetyl phosphate (40 pmoles), 4.3 moles of 14C- acetyl groups were bound per mole of enzyme. Since there are only 4.0 active cysteine sites per tetramer, this value indicates some dual labeling of both the cysteine and the lysine moiety of a given monomer (mol wt 35,000) (8). Table III also shows that p-nitrophenyl acetate was a more effective substrate for the acetylation reaction as only 2.0 pmoles were required to acetylate more than three active sites at 0”. Since p-nitrophenyl acetate is more reactive than acetyl phosphate, the acetylation of the enzyme with p-nitrophenyl acetate at room temperature tended to be less specific. Thus it has been easier to prepare the 14C- N-acetyl enzyme with acet.yl phosphate than with p-nitrophenyl acetate at pH 8.5 and room temperature.

In the presence of DPN, acetylation was markedly reduced. Only with the highest amount of acetyl phosphate (40 pmoles) was there significant labeling of the enzyme. As noted previ- ously, however, the low values could be explained by the rapid deacylation of the S-acetyl enzyme in the presence of DPN and by the competition between DPN and acetyl phosphate for lysine binding sites (7, 11).

The distribution of the acetyl group between the cysteine and Effect of Various Inhibitors on Acetylation of Dehydrogenase

TABLE III

E$ect of temperature, substrate concentration, and DPN on acetylation of S-phosphoglyceraldehyde dehydrogenase

DPN-free dehydrogenase (see “Materials and Methods”) was used with or without the addition of 10 eq of DPN. The enzyme

(0.1 pmole) was incubated in 0.1 M Tris, pH 7.0, at 0” or 25’with the designated amount of KLacetyl phosphate in a total volume of 1.2 ml. After 30 min, the number of bound I%-acetyl groups

was determined as described in Table I. The results of incuba- tion for 10 min with 2.0 pmoles of I%-p-nitrophenyl acetate are shown on the bottom line of the table.

Substrate

Acetyl phosphate

P;~j;y;’

acetate

pm&s

4 10 20 40

I 2

-

1

--

_-

GA&y1 groups bound per mole of DPN-free enzyme

‘4C-Acetyl groups bound per mole of enzyme-(DPN)a

00 2.50 00 250

moles moles

I 0.4 1.0 2.8 0.1

1.6 3.5 0.5 1.7 4.0 0.6 2.1 4.3 0.9 3.2 0.0

0.7

1.4

lysine moieties was also affected by the substrate concentration and the time of incubation. For example, at pH 7.0 and room temperature, the 14C-acetyl group was found predominantly in X-acetyl linkage when the enzyme was incubated with 40 eq (4.0 pmoles) of acetyl phosphate for 20 min. However, if the acetyl phosphate concentration and the time of incubation were both doubled, the 14C-acetyl label was then equally distributed between the cysteine and lysine moieties. Similar differences in the distribution of the acetyl group could also be shown with p-nitrophenyl acetate as substrate.

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Issue of June 10, 1970 J. H. Park, D. 6. Thaw, E. Mathew, and B. P. Meriwether 2949

'YABLE IV

Effeci of various inhibitors on acetylation of 3-phosphoglyceraldehyde dehydrogenase with 14C-acetyl phosphate OT

%-p-nitrophenyl acetate

The dialyzed, DPN-free enzyme (0.1 pmole) was previously

treated with the inhibitor in the molar ratios designated in the table and then incubated with acetyl phosphate (4.0 pmoles) for 30 min at 25”, pH 7.0 or pH 8.5. The acetyl phosphate concentra-

tion and incubation time were selected so that the acetylation at pH 7.0 would be primarily on the cysteine. With p-nitrophenyl acetate (2.0 pmoles) as the acetylating substrate, the reaction was

carried out for 15 min at 0”, pH 7.0. The volume was approxi- mat,ely 1.2 ml. The moles of I%-acetyl groups bound to the en- zyme were determined as described in Table I.

Additions

None......................

Iodoacetic acid. . p-Hydroxymercuribenzo-

ate ._ Iodosobenzoate L-1-Tosylamido-2-phenyl-

chloromethyl ketone..

Phenyl methane sulfonyl fluoride. . , . . . . . . . .

-

Molar ratio 31 inhibitor to enzyme

20

10

20

6

20 -

Inc$gion

inhibitor

min 30

30

10

10

30

30

GAcetyl groups mund per mole of DPN-free enzyme

MC-

fh;t”o UC-*- Nitro-

phate, phenyl pH 7.0 acetate, or 8.5 pH 7.0

moles

2.1

0.0

0.0

0.0

0.2

2.1

3.1 0.0

0.0

0.2

0.4

with ;Icetyl Phosphate and p-Nitrophenyl Acetate-Iodoacetic acid, p-hydroxymercuribenzoic acid, or iodosobenzoate blocked S-acetyl enzyme formation with acetyl phosphate or p-nitro- phenyl acetate at pH 7.0 (Table IV). In the case of acetyl phosphate, the sulfhydryl inhibitors are also effective at pH 8.5, at which the label was predominantly N-acetyl and not X-acetyl. A limited acetylation of the lysine moiety by p-nitrophenyl acetate at pH 8.5 has been shown with a DPN-free enzyme treated with iodosobenzoate or iodoacetic acid (8, 11). A similar experiment with high concentrations of acetyl phosphate to force the labeling of the lysine moiety of a carboxymethylated enzyme has not been attempted.

L-l-Tosylamido%phenylchloromethyl ketone (22) inhibited the acetylation of the enzyme with both acetyl phosphate and p-nitrophenyl acetate. Phenyl methane sulfonyl fluoride (23) did not affect the acetylation by acetyl phosphate.

Eflect of N-Acetylation on Binding of DPN to Enzyme-The effect of S- and N-acetylation on DPN binding was studied with several concentrations of acetyl phosphate at pH 4.5, 7.0, and 8.5 (Fig. 2). When 4 eq of DPN were added to the DPN-free enzyme in the absence of acetyl phosphate, 2.8 to 3.0 moles of coenzyme were bound per mole of enzyme. At pH 4.5, addition of acetyl phosphate had little effect on the DPN binding (Fig. 2) since the acetyl group was in thioester linkage (Fig. 1). At pH 7.0, however, there was an impairment of the DPN binding which increased as the concentration of acetyl phosphate was raised (Fig. 2). This inhibition was proportional to the extent of N-acetylation of the enzyme. The greatest reduction in

w 3,ot

i b x- pH 4.5

z 1.0’ ’ ’ ’ ’ ’ ’ c ’ ’

4 IO 20

/.IMOLES OF ACETYL PHOSPHATE

FIG. 2. The effect of N-acetylation on the binding of DPN to the dehydrogenase. DPN-free enzyme (0.1 pmole) was incubated for 30 min at pH 4.5,7.0, or 8.5 in the absence or presence of acetyl phosphate (4,10, or 20 pmoles) as indicated on the abscissa. Tem- perature, 25”. Then 4.0 eq of DPN were added and 10 min later the mixture was placed on a Sephadex G-25.fine column in order to separate the enzyme-DPN complex from the free DPN. The eluting buffers were 0.005 M Tris-0.001 M EDTA adjusted to pH 7.0 or 8.5. The amount of bound DPN was determined spectro- photometrically from the A280. *A,,, ratio of the eluted enzyme, and the free DPN was measured as a separately eluted peak. The sum of the bound and free DPN equaled the amount of DPN added initially.

binding occurs at pH 8.5, at which the enzyme was N-acetylated to the largest extent (Figs. 1 and 2).

The correspondence between the inhibition of the DPN bind- ing and N-acetylation was verified chemically, as shown in Table V. Samples of DPN-free enzyme were first incubated in the presence or absence of acetyl phosphate and then tested for DPN binding capacity at pH 7.0 and 8.5. The control enzymes with no added acetyl phosphate bound 2.9 moles of DPN. By comparison, at pH 7.0, the partially N-acetylated enzyme bound 2.0 moles of DPN per mole of enzyme, and at pH 8.5 only 1.3 moles of DPN were bound. At pH 7.0 the acetyl groups were about equally distributed between the cysteine and lysine moi- eties, but at pH 8.5 the acetyl group was predominately linked to the lysine residue.

The data on the last line of the table indicated that it was the N-acetylation and not the high pH of 8.5 that inhibited the bind- ing of DPN. The enzyme was first N-acetylated at pH 8.5 and the pH was dropped to 7.0 before the addition of DPN. It had been shown previously that lowering the pH to 7.0 did not remove acetyl groups from the lysine residues (11). The result was identical wit,h that of the preceding experiment at pH 8.5 as only 1.3 moles of DPN were bound per mole of enzyme.

The complete inhibition of DPN binding has not as yet been achieved. This could be explained by two observations. First, the theoretical number of N-acetylated sites (four per tetramer, mol wt 140,000) is rarely attained, as a result of partial oxidation of the reactive cysteine residues which are required for S-N transfer. Second, the DPN molecules are bound to the tetra- merit enzyme with different affinities and only the loosely bound coenzyme may be displaced (24-26).

In order to reinforce the conclusion that the -SH group at the active center is not essential for DPN binding, the DPN-free enzyme was carboxymethylated with iodoacetic acid at pH 7.0 or 8.5. Under these conditions only the reactive cysteine at the

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2950 N-Acetylation of S-Phosphoglyceraldehyde Dehydrogenase Vol. 245, No. 11

Relationship between S- and N-acetylation of

dehydrogenase and DPN binding

The DPN-free enzyme (0.1 pmole) was incubated in the absence and presence of 14C-acetyl phosphate (10 /Imoles) at pH 7.0 or 8.5 for 30 min in a total volume of 1.0 ml. Then 4.0 eq of DPN were

added to the enzyme samples and 10 min later the mixtures were placed on Sephadex G-25 fine columns in order to separate the enzyme-DPN complex from free DPN. The moles of bound DPN

were determined as described in Fig. 2. The I%-acetyl enzyme compounds were precipitated with an acetone-ether-HCl mixture, washed free of contaminating radioactivity, and air-dried. The total number of acetyl groups bound per mole of enzyme was

determined as described in Table I. A suitable portion of the air- dried material was oxidized with performic acid to remove the

S-acetyl groups, and the preparation was counted again to deter- mine the number of acetyl groups bound to the lysine in N-acetyl linkage. The last line of the table represents a separate experi- ment on coenzyme binding in which the enzyme was N-acetylated

at pH 8.5 and the pH was then lowered to 7.0 before the addit,ion of the DPN and passage of the enzyme through the Sephadex col- umn.

DPN bound per mole of enzyme

“C-A&y1 groups bound per

PH mole of enzyme

Total 1 S-Acetyl IN-A&y1

mazes mozcs 7.0 None 2.8

3.2 1.8 1.4 2.0

8.5 None 2.9 2.9 0.7 2.2 1.3

8.5 for acetylation and 3.0 1.3 then lowered to 7.0 for DPN binding and

Sephadex treatment L

active site is carboxymethylated (6). When the carboxymethyl- ated enzyme was treated with 4 eq of DPN and passed through a Sephadex column, 2.4 moles of bound DPN per mole of enzyme were found as compared to 2.8 moles in the native enzyme. Although carboxymethyl or acetyl groups attached to cysteine residues do not significantly affect the amount of bound DPN, the larger molecule, p-hydroxymercuribenzoate, does prevent DPN binding and inactivate the enzyme (27).

The DPN bound to native enzyme cannot be displaced to any significant extent by acetyl phosphate. When enzyme contain- ing 4 eq of bound DPN is incubated at pH 7.0 or 8.5 for 30 min

with 200 eq of acetyl phosphate, only 0.3 or 0.4 eq of DPN is displaced. Although there is a rapidly reversible acetylation of the reactive cysteine residue of the enzyme-(DPNjl complex as shown by 32P exchange reactions with acetyl phosphate (I), the N-acetylation of the enzyme is prevented by DPN.

Effect of N-Acetylation on Dehydrogenase Activity-Since N-

acetylation impairs DPN binding, it would be expected to in- hibit dehydrogenase activity. The extent of the inhibition is dependent on the particular conditions of the assay system such as the enzyme, DPN, or cysteine concentration. Moreover, the activity of the N-acetylated enzyme has been found to de- crease markedly over a 2-hour time period. Assays of the N-acetylated enzyme with DPN and time as the variables are shown in Fig. 3.

: 401 \ L N-ACETYL ENZYME 0 E \ t DPN a *

20

\*NN-ACETYL ENZYME DPN-FREE

0’ I I I 2

HOURS AFTER N-ACETYLATION

FIG. 3. The effect of N-acetylation on the dehydrogenase ac- tivity of the enzyme with DPN and time as variables. The en- zyme-(DPN)r complex and the charcoal-treated DPN-free enzyme were dialyzed as previously described. For controls, 0.1 pmole of each preparation was incubated in 1.0 ml of 0.1 M Tris buffer, pH 8.5, for 45 min at room temperature in the a.bsence of acetyl phos- phate. The enzymes were then diluted 1:400 in pyrophosphate- cysteine buffer and suitable aliquots were assayed for dehydrogen- ase activity in the conventional system (13) with 160 kmoles of pyrophosphate buffer, pH 8.5, 10 pmoles of cysteine, 40 pmoles of sodium arsenate, 1.2 pmoles of DPN, and 1.2 pmoles of 3-phos- phoglyceraldehyde. Total volume, 3.0 ml. The activity of the diluted enzyme-(DPN)a complex at zero time was taken as 100%. The 1:400 diluted enzyme preparations were stored at 0” and as- sayed again 1 and 2 hours later. X--X, enzyme-(DPN)r com- plex; A--A, DPN-free enzyme. To test the effects of acetyl phosphate (AC - P), the enzyme-(DPN)a complex and the DPN- free enzyme (0.1 kmole) were each incubated with 20 pmoles of acetyl phosphate in 1.0 ml of 0.1 M Tris buffer, pH 8.5, for 45 min at room temperature. Under these conditions only the DPN-free enzyme is N-acetylated. The enzymes were then diluted 1:400 and assayed in the same manner as the controls. O-O, en- zyme-(DPN)a complex with added acetyl phosphate; *p*, N-acetyl enzyme-DPN-free. In a separate experiment, 2.0~moles of DPN were added to 0.1 pmole of N-acetplated dehydrogenase; the mixture was diluted 1:400 and assayed as described above. O-O, N-acetyl enzyme with added DPN.

The control enzyme-(DPN)., complex lost no activity during a 45-min incubation in concentrated solution in Tris buffer at pH 8.5 and room temperature. When the control enzyme was then dihited 1:400 and stored at 0” for 2 hours, there was a 10% decline in activity (Fig. 3, top curve). Essentially the same re-

sults were obtained when the enzyme-(DPN)I complex was incubated with acetyl phosphate since the coenzyme protected against the N-acetylation (Fig. 3, second curve).

The DPN-free enzyme is susceptible to inactivation, and even in a concentrated solution (14 mg of protein per ml) the enzyme needs DPN to maintain full activity during the 45-min incuba-

tion at room temperature. Thus the initial assay of the DPN- free enzyme was about 10yO less than the control enzyme- (DPN)I complex (middle curve). After dilution and storage for 2 hours, it had lost 30% activity. N-Acetylation of the DPN- free enzyme made it even more unstable (bottom curve). The N-acetylated DPN-free enzyme was initially inhibited 20 to 30% and 2 hours after dilution it was virtually inactive. As shown, the addition of DPN to the N-acetylated enzyme afforded partial

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TABLE VI

Effects of DPN in stabilizing both charcoal-treated DPN-free dehydrogenase and N-acetylated enzyme

Five enzyme samples, each containing 0.1 pmole of the charcoal treated DPN-free dehydrogenase in 1.0 ml of 0.1 M Tris buffer, pH 8.5, were then treated in the following manner. Sample l- enzyme-(DPN)l complex: The DPN-free enzyme was reconsti- tuted by the addition of 1.0 pmole of DPN and then placed at room

temperature for 45 min. Sample 2-DPN-free enzyme: The en- zyme was placed at room temperature for 45 min. Sample 3- N-acetyl enzyme: The DPN-free enzyme was incubated with 20 pmoles of acetyl phosphate for 45 min at room temperature. Sam- ple 4--iV-acetyl enzyme with added DPN: The enzyme was N-acetylated with 20 pmoles of acetyl phosphate as described for Sample 3 and then 1.0 pmole of DPN was added. Sample 5-en- zyme-(DPN)( complex with added acetyl phosphate: The enzyme was reconstituted by adding 1.0 pmole of DPN and 5 min later 20 pmoles of acetyl phosphate. This enzyme was then incubated as described for Sample 2. All enzyme samples were placed at 0”

and then assayed immediately for dehydrogenase activity as out- lined in Fig. 3. The enzyme-(DPN)a complex (Sample 1) had the highest specific activity and was taken as loo’%. The samples

were kept in ice for 24 hours and then reassayed. The percentage activity was again calculated on the basis of the zero time assay of the enzyme-(DPN)d complex.

Type of enzyme sample

Dehydrogenase activity

zero time 24 hrs later

%

1. Enzyme-(DPN)d complex.

2. DPN-free enzyme. 3. N-Acetyl enzyme. 4. N-Acetyl enzyme + DPN.

5. Enzyme-(DPN)h + acetyl phos-

100 98

88 45 77 11 79 65

phate........................... 96 95

protection against the inactivation. None of the reagents used in the dehydrogenase assay could deacetylate the N-acetyl enzyme.

When acetyl phosphate was first added to DPN-free enzyme just before the assay, the activity of the enzyme was not in- hibited. On the other hand, when a concentrated solution of the DPN-free enzyme was incubated with the acetyl phosphate for 1, 2, and 3 hours, the percentage inhibition did not progress after the 1st hour. The factors determining the extent of in- hibition under these conditions are considered under “Discus- sion.”

The inhibition of the N-acetylated enzyme as shown in Fig. 3 is the minimum inhibition observed under various assay condi- tions. Inhibition of the N-acetyl enzyme could be increased in several ways. For example, omission of cysteine in the initial assay of the N-acetyl dehydrogenase produced a 40 to 80% inhibition instead of the 20% recorded in Fig. 3. Reduction of either the enzyme or the DPN concentration by one-fourth re- duced the activity of the N-acetyl enzyme to 40 to 60% of the control. Thus far, the substrate, 3-phosphoglyceraldehyde, had no protective effect.

These data suggest that the N-acetyl enzyme may be unstable and change its conformation in the absence of DPN or cysteine. In order to substantiate this possibility, another type of experi- ment was designed involving prolonged incubations of enzymes in concentrated solution. As shown in Table VI, the DPN-free

enzyme and the N-acetylated enzyme had 88 and 77% of the activity of the control enzyme-(DPN)r complex (Samples 1, 2 and 3). The addition of DPN to the N-acetylated enzyme (Sample 4) did not reactivate this dehydrogenase. However, when DPN was added to the enzyme before the acetyl phosphate, the coenzyme protected the enzyme against any inactivation (Sample 5). All five enzyme samples stood for 24 hours at 0” in concentrated solution (14 mg per ml) and were then reassayed. The enzyme-(DPn’)4 complex and the enzyme-(DPN)l complex with added acetyl phosphate retained full activity (Samples 1 and 5). The DPN-free enzyme (Sample 2) was only 45y0 as active as the control and the N-acetyl enzyme was almost com- pletely inactivated (Sample 3). The N-acetyl enzyme with added DPN (Sample 4) was partially protected.

A conformational change in the N-acetylated enzyme became apparent with the development of turbidity after standing 24 hours. After 48 hours, the N-acetyl enzyme had precipitated, the DPN-free enzyme had become turbid, but all tubes with added DPN remained completely clear.

DISCUSSION

Acetylation of triose phosphate dehydrogenase by acetyl phosphate has a high degree of specificity since no complex is formed in the case of aldolase, hexokinase, and albumin (Table I). Aldolase is noteworthy because it forms an enzyme-sub- strate compound with dihydroxyacetone phosphate and an active center lysine moiety (28) and, therefore, might have been sus- ceptible to N-acetylation with acetyl phosphate.

The 14C-acetyl-enzyme ratio of the complex formed with the muscle dehydrogenase and ‘4C-acetyl phosphate or p-nitrophenyl acetate under a variety of conditions is between 3.0 and 3.8 (4, 11). p-Nitrophenyl acetate is a more effective acetylating substrate than acetyl phosphate (Table III). Under the same conditions, the yeast dehydrogenase is not as readily acetylated with these substrates (5) as the muscle enzyme. The acetyl to enzyme ratio of the complex formed with the yeast enzyme is only 1.2 with acetyl phosphate and 2.2 to 2.9 with p-nitrophenyl acetate. Again p-nitrophenyl acetate is a more efficient acetylat- ing compound.

The sulfhydryl inhibitors, iodoacetic acid, iodosobenzoic acid, and p-hydroxymercuribenzoic acid, prevent both the S- and the N-acetylation reactions with acetyl phosphate (Table IV). These results are in accord with the finding that S--N transfer is the major route for the N-acetylation of the enzyme (7, 8). There is a small amount of direct N-acetylation when the more reactive substrate, p-nitrophenyl acetate, is incubated with a carboxymethylated enzyme (8). An N-acetylated enzyme can be readily carboxymethylated with iodoacetic acid (8) or slowly acetylated in the presence of high concentration (0.2 M) of sub- strates.

L-1-Tosylamido-2-phenylchloromethyl ketone also blocks S- and N-acetylation of the enzyme. Although this reagent reacts with histidine residues of chymotrypsin (22), the site of interaction with the dehydrogenase has not been determined. Histidine has been implicated by a variety of methods as a participant in the various activities of the dehydrogenase (29-32). However, the inhibitor could also react with the sulfhydryl group in the active center octadecapeptide of the dehydrogenase.

Phenyl methane sulfonyl fluoride was without effect on the acetylation reactions. The result is in accord with the earlier finding that the dehydrogenase is not inhibited by diisopropyl-

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2952 N-Acetylation of 3-Phosphoglyceraldehyde Dehydrogenase Vol. 245, No. Ii

fluorophosphate except in excessively high concentrations (4). These data suggest that serine does not directly participate in the acetglation reactions. However, the serines in the active center may in part provide a hydrophilic environment necessary for the over-all oxidation reaction which involves negatively charged substrates, coenzymes, and inorganic phosphate.

Although the sulfhydryl groups at the active sites are not essential for DPN binding, the reactive lysine residues are re- quired for maximal coenzyme binding. The elegant sequence work of Harris and Perham (33) showed that the active site cyst)eine is at position I49 and the reactive lysine at position 183 in the monomer containing 332 amino acids. Although cysteine- 149 and lysine-183 are not near neighbors in the primary se- quence, these residues are closely approximated by the folding of the peptide chains as shown in the S-N transfer reaction. It is reasonable that cysteine-149, which participates in thioester and thiohemiacetal linkages with substrates, should not be re- quired for DPN binding. However, a charged lysine residue in the catalytic center could be readily involved in coenzyme bind- ing.

The quantitative data in Fig. 2 and Table V show that the control enzyme binds 2.8 to 3.0 of the 4.0 added eq of DPN after passage through the Sephadex column. Since the coenzyme is dissociable and in equilibrium with the free DPN, the enzyme cannot retain the full complement of 4 moles of DPN after the Sephadex treatment. N-Acetylation of the enzyme reduces the number of bound DPN molecules to 1.2 eq. Initial attempts to inhibit completely the DPN binding by prolonged incubation with excess acetyl phosphate have not been successful. There are two explanations why approximately 1 mole of DPN per mole of enzyme is resistant to displacement from the enzyme by AT-acetylation.

First, tightly bound DPN could be associated with a monomer which is not N-acetylated. Generally, the ratio of moles of acetyl groups bound per mole of enzyme ranges from 3.0 to 3.5. The incomplete acetylation of the tetramer with acetyl phosphate is presumably due t,o the fact that some of the very reactive sulfhydryl groups at the active site are partially oxidized and the full complement of cysteine residues is not available for acetyla- tion. However, an active center in which the sulfhydryl groups are oxidized can still bind DPN. This was verified by the fact that DPN is bound to an enzyme treated with iodosobenzoate ox inactivated by incubation in the absence of reducing reagents. Thus, a nonacetylated and partially oxidized active center could bind 1 eq of coenzyme.

Second, an alternative explanation for the residual DPN bind- ing of the N-acetylated enzyme derives the fact that some DPN molecules may be bound more tightly than others (34-36). In the muscle enzyme, there appear to be 2 tightly bound DPN molecules with dissociation constants between lo+ and lop9 M

and two loosely bound coenzymes with dissociation constants between 3 x 1O-7 and 2 x lop5 M (35). The tightly bound DPN may not be affected by N-acetylation, whereas the loosely bound coenzyme could be displaced by acetyl phosphate.

Polgar has been unable to confirm the inhibition of DPN bind- ing by N-acetylation of the enzyme (37). The reasons for this discrepancy are probably differences in experimental conditions. In Polgar’s study the dehydrogenase from pig muscle was acety- lated with p-nitrophenyl acetate at pH 8.0 and room tempera- ture, and the molar ratio of acetyl groups to enzyme was 2.1. Under these conditions with the rabbit enzyme we have found

that p-nitrophenyl acetate tends to give some nonspecific label- ing of amino and sulfhydryl groups. Starting with a ratio as low as 2.1, there may actually be rather incomplete acetylation of the specific lysine residues and therefore minimal effect on DPN binding. In the case of Polgar’s experiments, the extent of DPN binding was also measured after passage of the enzyme- DPN complex through a Sephadex column. The amount of DPK bound was calculated only from the spectrophotometric reading of the Azso :AsGO ratio of the enzyme-DPN complex and free DPN was not determined. Since any turbidity of the en- zyme would lower the A ZsO:il 260 ratio, the balance of free DPN needs to be considered. Such factors may well account for the differences in the results from the two laboratories.

The dehydrogenase activity of the N-acetylated enzyme is inhibited to varying degrees depending on the assay conditions. This inhibition is due in part to the instability of the N-acety- lated enzyme. Under conditions which cause inactivation, i.e., high dilution and prolonged standing in the absence of cysteine, there is a rapid inactivation and eventual precipitation of the N-acetyl enzyme. Since this inactivation can be partially pre- vented by added DPN, it appears that the coenzyme may be protecting the enzyme against a conformational change. In this regard, the work of Listowsky et al. (38) is interesting as they showed that 1 mole of DPN per mole of tetramer produced the major conformational change in the conversion of the apoenzyme to the enzymatically active dehydrogenase. Since 1 mole of DPN is bound to the N-acetylated tetramer (Fig. 2 and Table V), it is possible that this small complement of bound coenzyme may protect the enzyme against inactivation and precipitation (Fig. 3 and Table VI).

In the assay of the dehydrogenase activity, the percentage inhibition of the N-acetylated enzyme is related to the concentra- tion of the DPN. At 4 x lop4 M DPN there is 20% inhibition and at 1 x lo+ M DPN 40 to 60% inhibition. The increase in inhibition at the lower DPN concentration could be related to the protective effect of the coenzyme noted in the preceding paragraph. In addition, there is also a competition between acetyl phosphate and DPN for occupancy in the active center (Table III). Therefore, a higher concentration of DPN would promote a better rate of catalysis.

There is no direct correlation between the 60% inhibition of DPN binding (Fig. 2 and Table V) and the 25% inhibition of the dehydrogenase activity (Fig. 3 and Table VI). The reason for lack of the correlation is due to the differences in the testing conditions. For example, in the DPN binding experiment no cysteine was added because the cysteine can act as an acceptor for the acetyl group of the S-acetyl enzyme complex and thereby deacylate the enzyme. In the dehydrogenase assay, however, cysteine is present in order to promote maximal rates of oxida- tion of 3-phosphoglyceraldehyde. The absence of cysteine in the DPN binding experiment means that any oxidation of sulf- hydryl groups at the active center prevents the S--N transfer reaction necessary for N-acetylation but does not inhibit DPN binding. When cysteine is added to the N-acetylated enzyme in the dehydrogenase assay, the oxidized sulfhydryl groups are reduced and catalyze the oxidation of the substrate. Since the oxidized active centers cannot be N-acetylated but can be acti- vated for the dehydrogenase reaction by the added cysteine, the possibility of correlating the N-acetylation, DPN binding, and dehydrogenase inhibition is difficult. When the N-acetylated enzyme is assayed in the absence of cysteine, the 60 to 80%

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Issue of June 10, 1970 J. H. Park, D. C. Xhaw, E. Mathew, and B. P. Meriwether 2953

inhibition of DPN binding is more closely related to the 50 to 80% inhibition of dehydrogenase activity. In this case, only those reduced active centers which participate in the S-N transfer reaction are measured in the dehydrogenase assay. Although the quantitative aspects of DPN binding and dehydrogenase inhibition would be improved with a completely reduced N- acetylated enzyme, there is nonetheless an obvious interrelation- ship between N-acetylation, DPN binding, conformation, and enzyme activity.

Since N-acetylation inactivates the enzyme, the biological consequences of this inhibition should be considered. It appears as if the enzyme has a built-in defense against this type of in- activation by virtue of its coenzyme which prevents N-acetyla-

tion. It is interesting in this regard that 3-phosphoglyceralde- hyde dehydrogenase, which binds DPN very tightly, is unique among dehydrogenases in having a higher binding constant for DPN than DPNH and crystallizing with 3 moles of DPN bound per mole of enzyme (14). In yeast cells, the spectrophotometric measurements indicate that DPN is bound to the enzyme in

vivo (39). The DPN concentration in many types of cells is about 15 times greater than the DPNH. The equilibrium con- stants and biological conditions are such that when the enzyme- bound DPN is reduced by oxidation of 3-phosphoglyceraldehyde to 1,3-diphosphoglyceric acid the newly formed DPNH is readily replaced by DPN. The enzyme is thereby well protected in the cellular cytoplasm. Thus DPN may have the unexpected role of properly orienting the substrates at the active site and forestalling any acylation of “incorrect moieties” on the enzyme.

Acknowledgments-We wish to thank Drs. Charles R. Park and Sidney P. Colowick for their helpful suggestions in the course of this work.

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Jane Harting Park, Denis C. Shaw, Elizabeth Mathew and Blanche P. MeriwetherDehydrogenase by Acetyl Phosphate

-Acetylation of 3-PhosphoglyceraldehydeNEnzymatic Characterization of the

1970, 245:2946-2953.J. Biol. Chem. 

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