Trrr: JOURNAL OF BIOLOQICAL CHEMIYTRY Vol. 234, No. 7. July 1959

Printed in U.S.A.

Structure of the Site Phosphorylated in the

Phosphorylase b to a Reaction*?

EDMOND H. FISCHER, DOXALD J. GRAVES,~ ELOISE R. SSYDER CRITTENDEN, AND EDYIN G. KREBS

From the Department of Biochemistry, University of Washington, Seattle, Washington

(Received for publication, January 14, 1959)

In previous publications from this laboratory (2-4), it was re- ported that the conversion of phosphorylase 6 to phosphorylase a, in the presence of big++, ATP, and phosphorylase b kinase, resulted in the incorporation of 4 moles of phosphate per mole of phosphorylase a, according to Reaction 1 (4):

phosphorylase b

2 phosphorylase b + 1ATP kinase

, Mn++ or Mg++

phosphorylase a + 4ADP (1)

In order to investigate the mode of binding of the phosphate groups to the protein molecule, P32-labeled phosphorylase a was prepared with the use of ATP32. The labeled enzyme was then subjected to a tryptic attack and the radioactive phosphopep- tides released were isolated according to conventional procedures. Three pure, closely related phosphopeptides were obtained and their structure and amino acid sequence determined.

EXPERIMENTAL

Materials

Crystalline P32-labeled phosphorylase a was prepared as de- scribed previously (4), with the use of crystalline phosphorylase 6 (5), ;\lg++, phosphorylase b kinase, and purified Paz-labeled ATPI (6,7). The labeled phosphorylase a produced was purified by precipitation with ammonium sulfate and crystallized as de- scribed (4). Trypsin and chymotrypsin were purchased from the Worthington Biochemical Corporation as twice crystallized ma- terials containing approximately 50 per cent &lgSOI. They were dialyzed overnight in the cold against dilute HCl, pH 3.0, and kept frozen at this pH. Immediately before use, their solutions were neutralized to pH 7.0 with 1O-3 N NaOH. Carboxypeptidase A, twice crystallized,* was a generous gift from Rliss Barbara

* Supported by a research grant (A-859) from the National In- stitutes of Health, United States Public Health Service.

t A preliminary account of this work has been reported (1). $ Predoctoral fellow of the National Institute of Arthritis and

Metabolic Diseases, United States Public Health Service. 1 Where large amounts of P32-labeled phosphorylase a were

needed, the conversion of phosphorylase b to a was carried out directlv with a crude ATPa solution obtained from the oxidative phospgorylation incubation mixture described by Kielley and Kiellev (6) rather than with nurified ATP32. The crude incuba- tion ria&ion mixture is used after perchloric acid precipitation and neutralization with KOH.

2 Carboxypeptidase A was prepared from an acetone powder of beef pancreas glands by a procedure developed in Dr. H. Neu- rath’s laboratory.

Allen. Carboxypeptidase B was prepared by the method of Folk and Gladner (8) and was generously supplied by Dr. J.-F. Pechere. Leucyl-aminopeptidase was purified according to the procedure of Smith et al. (9), omitting the final starch zone-elec- trophoresis step.

Methods

Control of Tryptic Attack of P32-labeled Phosphorylase a-In the

experiment designed to control the release of bound phosphate from phosphorylase a, neutralized trypsin was added in various amounts to a solution of 4.7 mg. of PWabeled phosphorylase a (3530 c.p.m. per mg.), pH 7.5, bringing the final volume of the reaction mixture to 0.8 ml. At various times O.l-ml. samples were removed, added to graduated centrifuge tubes containing 1.0 ml. of a cold solution of carrier albumin (2.5 mg. per ml.), and the mixtures were immediately precipitated by addition of an equal volume of 10 per cent trichloroacetic acid. The sus- pensions were centrifuged after standing for 15 minutes at 0”. Aliquots of the supernatant solutions were plated and counted for determination of the radioactivity released from the protein. As a control, if needed, the pellets were also taken up in 80 per cent formic acid to a volume of 1.0 ml. and aliquots were plated, dried at moderate heat under a stream of air, and counted.

Preparative Tryptic Attack of PWabeled Phosphorylase a-P32-

phosphorylase a, 200 to 300 ml., (approximately 25 mg. per ml.), treated with cysteine and freed from AMP as described under “Results,” pH 7.2 to 7.5, 25’, is mixed with an equal volume of a neutralized solution of trypsin. A 1 to 1 molar ratio of trypsin to phosphorylase a is used, corresponding approximately to a l/20 weight ratio. After 1.0 to 1.5 minutes, the reaction is blocked by addition of 0.1 volume of cold 50 per cent trichlo- roacetic acid and the suspension is cooled in ice for 15 to 20 minutes. This suspension is bright yellow at first, but turns colorless due to the fact that pyridoxal5’-phosphate, present in phosphorylase as a substituted aldamine (10, 11)) is transformed to a hydrogen-bonded Schiff base before being hydrolyzed. The suspension is centrifuged, the precipitate is washed twice with 50 ml. of cold 3 per cent trichloroacetic acid, and recentri- fuged; the combined supernatant solutions are extracted five times with an equal volume of peroxide-free ether and freeze- dried.

Ion Exchange Chromatography of P32-labeled Peptides-The orange colored residue obtained above is taken up in approxi- mately 25 ml. of water, the solution is adjusted to pH 3.0 with HCl, and chromatographed according to the procedure of Hirs et al. (12)) slightly modified in that the Dowex 50-X2, 200 to 400

1698

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mesh resin used in the operation is thoroughly washed with IO-3 M acetic acid, after being charged with the 0.2 M Na-citrate buffer, pH 3.1 (13). Once the material has been introduced in the col- umn (0.9 X 120 cm.), the latter is washed with 1O-3 M acetic acid until a small, fast moving radioactive Fraction A (see Fig. 3 be- low) has been eluted (approximately 200 ml.). By this pro- cedure, Fraction A is obtained directly free from salts. The bulk of the radioactive material, representing over 95 per cent of the total, remains absorbed at the top of the column. It is eluted with the 0.2 M citrate buffer, pH 3.1, then starting with Fraction SO, by gradient elution obtained by introducing 1 M citrate-ace- tate buffer, pH 5.1, (13) to a mixing flask containing 250 ml. of the pH 3.1 buffer (0.5 per cent BRIJ from the Atlas Powder Company is added to the two buffers). The flow rate is adjusted at about 1 drop per 15 seconds (5 minutes per ml.) for the salt elution; fractions of 250 drops corresponding to approximately 10 ml. are collected.

The three radioactive Fractions A, B, and C are collected; A is freeze-dried without further treatment while B and C are first desalted by the procedure of Haugaard and Haugaard (14), with the use of Dowex 50-X4, 100 to 200 mesh.

High Voltage Paper Electrophoresis and Paper Chromatography -Paper electrophoresis was carried out according to the pro- cedure of Ryle et al. (15), in pyridine acetate buffers pH 3.6 and 6.5, usually for 1 hour at 2000 volts. Sheets of Whatman No. 3 paper, 20 x 60 cm., were used, on which up to 3 pmoles of radio- active peptides could be streaked at a time. Preparative paper chromatography of peptides was carried out on Whatman h’o. 3 paper in the butanol:acetic acid :water solvent (4: 1:5). After the runs, the papers were dried and the radioactive bands, identi- fied by radioautography with Eastman Kodak “no screen” x-ray film, were cut out and eluted downwards with lvater.3 The eluates were freeze-dried.

Hydrolysis of Peptides for Anlino Acid Analysis-To samples of 5 to 25 ~1. containing 0.05 to 0.1 pmole of peptides, 0.3 ml. of twice redistilled, azeotropic 5.7 h’ HCl was added. The solu- tions were frozen in a dry ice-acetone mixture, and the tubes sealed under vacuum. Hydrolyses were then carried out by placing the scaled tubes in the vapor of refluxing isobutanol (107”). Hydrolysis was usuttlly complete after 16 to 24 hours. The tubes were cooled before opening, frozen in a dry ice-acetone mixture, and dried in an oil pump vacuum over NaOH.

Quantitative Amino Acid gnalysis-The amino acids present in the 12-, 16-, and 32hour hydrolysates were separated by chro- matography in the butanol-acetic acid solvent, quantitLttively eluted from the paper, and analyzed by the ninhydrin method (16). The amino end group analyses were performed with the use of the FDB4 method of Sanger (17), as given by Fraenkel- Conrat et al. (18). The dinitrophenyl amino acids were identi- fied by paper chromatography in the butanol-acetic acid and the the tertiary amyl alcohol-phthalate systems (18). The carboxy end group analyses were performed by the use of carboxypeptidase A and B.

Determination of Amide Sitroge?a-The method of Conway and Byrne (19) as applied by Wilcox et al. (20) was used. Glutamine and ammonium sulfate were used as standards in both 2 N NaOH and 0.05 M borate buffer, pH 9.7. The difference in amounts of

3 For the elution of peptides, low3 RI ammonia was more effec- tive than water and was generally used.

4 The abbreviations used are: FDR, fluorodinitrobenzene; DFP, diisopropylphosphorofluoridate.

ammonia liberated in these two test systems in 16 hours at room temperat.ure was found to be a mcasurc of the total amide nitro- gen present. The ammonia was determined by the calorimetric method of Moore and Stein (21).

RESULTS

Acid and Base Hydrolysis of Pa”-labeled Phosphorylase a-The acid-base lability of Pa2 in phosphorylasc a was investigated in order to determine the nature of the phosphate bond. It is well known that O-P bonds in proteins are labile in base but stable in acid (22). Although it has never been demonstrated, the con- trary can be assumed to be true for N-P bonds in proteins.

HCl or NaOH was added to crystalline suspensions of phos- phorylase a at 30” to a final concentration of 0.25 M. At various intervals of time samples were removed and neutralized. The denatured protein was removed by centrifugation, and the super- natant fluid was analyzed for inorganic Paz. High voltage paper electrophoresis, by which inorganic P can be effectively identified, indicated that only inorganic orthophosphate was released. Fig. 1 shows that the phosphate bond was labile in base but stable in acid, which is characteristic of an O-P bond such as would bc expected for a peptide containing a serglphosphate residue.

To check the possibility that an N-P to O-P transfer might precede hydrolysis of the bound phosphate, a hydrolysis was also carried out in 5.8 M urea which might be expected to interfere with such a transfer reaction (23). No change was observed in the stability of the phosphate bond under these conditions.

Tryptic Attack of P32-ZabeZed Phosphorylase a---When phos- phorylase b (24) is converted to phosphorylase a (25) through phosphorylation by ATP, Rig++, and phosphorylasc b kinnse, it loses its absolute AMP requirement. Two enzymes have been described that can convert phosphorylase a back to a form show- ing a complete requirement for AMP, namely, PR enzyme (26, 27) and trypsin (26, 28). The actions of these two enzymes on P32-1abeled phosphorylase a were investigated with the hope that they might provide some information on the mode of bind- ing of the phosphate group to the phosphorylase molecule and the site actually involved in this binding. PR enzyme was found to be unsuitable for this study, as it catalyzes the hydroly- sis of the bound phosphate (Reaction 2) acting, therefore, as a specific protein phosphatase (1). Trypsin, however, was found to liberate the bound phosphate in the form of a nontrichloro- acetic acid-precipitable organic derivative, according to Reac- tion 3:

Paz-phosphorylase a PR enzyme

(2‘) ‘-’ 2 phosphorylase b + inorganic P32

4 8 12 16 20 ‘24 HOURS

FIG. 1. $cid and base hydrolysis of Paz-labeled phosphorylase a. The liberation of P32 in 0.25 M HCl and 0.25 IVI NaOH at 30” is plotted against time.

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1700 Phosphopepticle from Phosphorglase a Vol. 234, hTo. 7

Pa*-phosphorylase a trypsin

. 2 phosphorylase b’ + organic 1’3% + nonlabeled peptides

Although phosphorylase b and b’ produced in these reactions are both enzymatically active when tested in the presence of AMP, they differ in that only phosphorylase b, produced in the PR en- zyme-catalyzed reaction, can be reconverted back to phosphoryl- ase a according to Reaction 1.

In order to determine the optimal conditions under which maximal release of the bound labeled phosphate could be achieved, for a minimal over-all degradation of the enzyme, the factors affecting the tryptic attack of phosphorylase a were in- vestigated. AMP was found to inhibit the tryptic attack and, therefore, phosphorylase a to be used in the reaction was subse- quently freed from this nucleotide. Addition of 0.01 M cysteine directly to the reaction mixture resulted in a considerable increase in the over-all breakdown of the protein molecule, and was avoided. On the other hand, tryptic degradation of an enzyme preparation which had stood for 2 to 3 weeks in the cold before use proceeded poorly, but the rate of release of P32 could be re- stored by a pretreatment of the enzyme with cysteine. The pro- cedure for the preparation of phosphorylase a preceding the attack which appeared to give the best results consisted of dis- solving phosphorylase a crystals in 0.03 M neutralized cysteine at room temperature (final concentration of protein between 10 and 20 mg. protein per ml.), passing the solution through a charcoal column (5), and dialyzing the AMP-free enzyme overnight against water brought to pH 7.5 by NHaOH to get rid of the cysteine.

The rate of release of bound P32 was measured at various con- centrations of trypsin (Fig. 2). As can be seen, there is a sudden release of nontrichloroacetic acid-precipitable counts followed by an appreciable slowing down of the reaction rate. In the first rapid phase of the reaction at high trypsin to phosphorylase a ratios, over 90 per cent of the bound P32 can be released while 95 per cent of the protein still precipitates in trichloroacetic acid.

puri$cution of PWabeled Phosphopeptides-The radioactive, trichloroacetic acid-soluble material released from a preparative tryptic attack of phosphorylase a was first purified by column

4 *A

10 20 30 MIN. 6 Peptide C, in the final chromatographic step, often appears as FIG. 2. Release of trichloroacetic acid-soluble P32-labeled ma- though it might consist of two distinct radioactive bands mi-

terial from phosphorylase a at various concentrations of trypsin. grating extremely close to one another. Separate elution of each Experiment carried out, at 25”, as indicated under “Methods.” of these two bands gave materials which could not be distinguished The molar ratios of trypsin to phosphorylase were: Curve A, con- from one another by amino acid analysis, end-group analysis, and trol with no trypsin; CPUW B, 0.165; Curve C, 0.33; Curve D, 0.66; behavior in subsequent enzymatic attack, so that they were rou- Cwve E, 1.32. tinely eluted together.

- 0. D. I C.PM

80 2.0 $

60 % pr c

z 1.0 4OQ

s 20 %

20 40 60 8p ID0 120 140 160 < l 10-3 M 0.2 M CITRATE BUFFER

1 M CITRATE-ACETATE fractions ACETIC A. pH 3.1 BUFFER pH 5.1

FIG. 3. Chromatography of t.richloroacetic acid-soluble ma- terial obtained from tryptic degradation of PWabeled phosphor- ylase a. The separation is obtained on a 0.9 X 120-cm. column of Dowex 50-X2. On the left, ordinate, optical density at 570 rnti for each effluent fraction after treatment with ninhydrin reagent is plotted. On the right ordinate, counts per minute are plot- ted. No corrections for base-line absorption have been applied.

chromatography as described under “Methods,” and the radio- active Fractions A, B, and C (Fig. 3) were collected.

On paper electrophoresis at pH 6.5, Fraction C showed the presence of one major radioactive band, and two minor radioac- tive bands representing approximately 1 per cent of the total radioactivity in Fraction C. These minor bands were identified as peptides A and B by their electrophoretic mobilities at pH 3.6 and 6.5, and their RF values on chromatography in the butanol- acetic acid system. In addition, as expected, several nonradio- active peptides were also present in Fraction C. Only the three radioactive bands were eluted for further purification (the minor peptides A and B were added to Fractions A and B). The final purification was carried out by large scale paper chromatography in the butanol-acetic acid system which served to remove any remaining peptide contaminants still present along with the ra- dioactive peptide C. 5

Fractions A and B were also further purified by paper electro- phoresis followed by paper chromatography, to obtain the pure radioactive peptides A and B, respectively.

Table I summarizes the percentage recovery and distribution of the various radioactive fractions in a typical preparative run.

Properties of Purified Radioactive Peptides-The peptides from the three fractions obtained as described above give a single radioactive and ninhydrin-staining spot (except peptide A) when analyzed by paper electrophoresis at pH 3.6 and 6.5, and by paper chromatography in the butanol-acetic acid solvent. Their properties are listed in Table II.

Quantitative analysis on three different preparations of purified peptide C indicated a 1 to 1 ratio of each amino acid to P32, with the following average values: Lys, 0.97; Glu, 0.94; Ileu, 1.08; Ser, 1.05; Val, 0.91; Arg, 1.03.

Genealogy of Peptides A, B, and C-The preceding results seemed to indicate that the first phosphopeptide released in the tryptic attack is a hexapeptide, which is transformed to two la-

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beled pentupcptidcs, very closely related in that they give the same 5 amino acids after hydrolysis, but differing in the fact that one of them (peptidc B) is neutral at pH 6.5 and ninhydrin posi- tive, whereas the second (peptidc A) is acidic under the same conditions and usually does not react with ninhydrin. In order to establish the exact relationship among these peptides, IWa- brlcd phosphorylase a was subjected to a prolonged tryptic at- tack. For the first 2 minutes, the reaction was run at 0” to slow down the initial phase. At various times, samples were with- drawn from the incubation reaction mixture, rapidly added to small test tubes placed in advance in a boiling water bath to block the reaction, and spotted on paper for elrctrophorctic sep- aration.

It can be seen from Fig. 4 that the first sample, removed and boiled immediately after adding trypsin to phosphorylase a, con- tains nearly all of its radioactivity in the form of peptidc C, al- though a definite amount of peptidc A was also present. As protcolysis proceeds, more pcptide A appears while peptide C disappears, but contrary to what was expected, at no time was there any real indication of the formation of peptide B. The only difference in procedure which could have accounted for the discrepancy between this experiment and the preparative tryptic attack from Ivhich peptide B was originally isolated was that, in the latter case, the tryptic attack was stopped by precipitating the proteins with trichloroacetic acid rather than by boiling. To investigate the effect of heating on the formation of the pep- tides, the 27-hour hydrolgsato was separated into two portions, and one was boiled to stop the reaction while the other receivrd 1O-4 JI 11FP to inhibit the trypsin. As can be seen by compar- ing these two last samples, in the absence of heating, 70 per cent of the radioactivity is in the form of peptide B. When the sam- ple is heated, pcptide B disappears and peptide A is formed.

In Fig. 5, the relative amounts of peptides (A + B) and C formed in the tryptic digest are plotted against time. It can be seen that under the conditions used in this experiment, within the first 10 seconds, two very fast reactions (called Tr I and Tr I’) take place, releasing all the radioactivity from phosphorylase a in the proportion of 70 per cent as peptidc C (Tr I) and 30 per cent as peptidcs A and B (Tr I’). In a subsequent, much slower reaction (called Tr II), peptide C is itself converted to peptides (A + 13). From this and similar experiments, it was calculated that the approximate relative rates of these enzymatic reactions arc Tr 1:Tr 1’:Tr II = 4: 1: < 0.002. The difference between the rates of formation of peptide B from the protein directly and from pcptide C is another example of how proteolysis of a pcptide bond can be affected by neighboring groups.

The relationship between peptides A, B, and C can be sum- marized as follows:

c Tr 17 \Tr II

/ L Tr I’ phosphorylase a~ B

nonenzymatic heat

>A

Considering the charge of the six amino acids and the phosphate group present in peptidc C, this phosphorylated hexapeptide should behave as a neutral compound around pH 6, whereas it clearly migrates towards the negative pole. Likewise, pcptide B, which should be acidic, behaves as a neutral compound but acquires its expected acidic character when converted to peptide A. This discrepancy could best be explained if one assumed

TABLE I

IZecoveTies of P-labeled peptides fTom tr!yptic attack of phosphoT!/lase a

Recoveries are given for a typical purification of 360 ml. of pure, AMP-free phosphorylase a solution containing 5.2 gm. of protein (10.5 pmoles) and 78.5 X lo7 c.p.m. (specific radioactivity of Ps2: 1.87 X 107 c.p.m. per pmoles l’, corresponding to a total of 42 p- moles 1’).

Fractions

Original 1’32.labeled phosphorylase a. Trichloroacetic acid supernatant so-

lution after tryptic attack. Fractions collected after Dowex 50

colnmn chromatography*. Fractions elated after high voltage

paper electrophoresist.. Fractions eluted after paper chroma-

tography$.........................

-

L

Total recovery of radioactive material in each fraction

C.#.IE.jlO~

78.5

71.5

64.7

37.6

29.0

42

38.2

34.7

20.2

15.5

91

s2.5

‘IS.0

37.0

* The distribution of radioactivity in the various fractions was as follows: A, 8 per cent; B, 14 per cent; C, 78 per cent.

t The low yield obtained for this step (58 per cent) results from the fact that (a) the desalting of the fractions obtained from col- umn chromatography is included in it, and (5) in cutting out t,he radioactive bands to be eluted, purity rather than high yields was desired.

$ The final distrihution of radioactivity in the various fractions was as follows: A, 14 per cent; B, 4 per cent; C, 82 per cent. The increasein peptide A at the expense of peptide B is due to the con- version of peptide B to A which will be discussed later.

TABLE II

Properties of puri$ecl phosphopeptides A, B, and C’

Properties

Kinhydrin reaction Electrophoretic

migration on pa- per, pH 6.5, at 2000 volts, 35 ma. in 1 hour (in cm.)*

Approximate RF’S in butanol-acetic acid-water, 4:1:5

Amino acids pres- ent after hy- drolysis

I- Peptide A Peptide R

Sone

+6 to 7

0.5 Glu Ileu.

Ser.Val.Arg

- +

0

0.4 Glu Ilen.

Ser .Val. Arg

Peptide C

+

-5

0.2 Lys. (;lu. Ileu. Ser. ‘i-al. Arg

* These values have been corrected for migration due to electro- osmosis.

that the glutamic acid residue of the hcxapcptide is present as glutamine.

A determination of amide nitrogen was carried out on two dif- ferent samples of peptide C and indicated a release of over 0.7 equivalent of ammonia, under conditions where glutamine was

6 The amide group could not rest on arginine, as it would have protected this COOH-terminal group from carboxypeptidase B (see next section), and the phosphate group would have to oe entirely substituted to account for the observed difference in charge.

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1702 Phosphopeptide from Phosphorylase a Vol. 234, No. 7

FIG. 4. Radioautogram of a paperelect,rophoretic separation of P3*-labeled peptides formed during a tryptic at,tack of phosphorylase a. The digestion reaction mixture contained 2.6 mg./ml. of phosphorylaxe a (52,000 c.p.m./mg.) and 1.2 mg. of trypsin, at pH 7.5. The temperature was 0” for the first 2 minutes of digestion, then 25”. At various times, 0.1..ml. samples lvere removed, boiled (except one of the 27-hour samples which received lo-* BI DFP to block the reaction, indicated as “non-heated”) and spotted on the paper.

elertrophoresis XV& run for 1 hour at 2000 volts, 35 ma., pH 6.5. The standard marker contained pure peptides A, B, and C.

MINUTES

A 13

FIG. 5. .4, rate of transformation of radiopeptides in a prolonged tryptic attack of 1’32-labeled phosphorylase a. The percentages of radioactive peptides were calculated from densitometric plots obtained from a radioautogram of the complete experiment il- lustrated in Fig. 3, using a Spinco “Analyt’rol” densitometer. B, rate of disappearance of pept,ide C plotted on a semilogarithmic scale.

quantitatively deamidated. The migration of purifitad peptide C in two close but distinct bands in the butanol-acetic chroma- tography mrntioncd earlier (footnote 5) might bc due to a partial dcamidation of this frat+ion occurring in previous purification strps.

The transformation of peptide B to pcptidc A could occur if prptidc B contained an amino-terminal glutaminyl residue. Heating of this peptide would result in the formation of a pyr- rolidone ring concomitant with deamidation. This in turn would account for the loss of reactivity of peptide A \vith ninhydrin and FDB (see below). This behavior of amino-terminal glutaminyl residues has been repeatedly obscrvcd (29, 30).

Stepwise Enzymatic Degradation and Amino Acid Sequence of Peptide C-The purified phosphorylated hexapcptidc in amounts ranging from 0.5 to 2.0 pmoles was subjcctcd to the action of

various mzymes, so that its complete structure could be deter- mintd. After each attack, the reaction mixture was subjected to paper electrophorcsis. Each radioactive band appearing on the elcctrophorctogram (as detected by radioautography) was elut.ed, furthrr purified by paper chromatography, then analyzed for total amino acids and for N-terminal amino acid. In addi- tion, all other ninhydrin-staining materials released in the attack were analyzed for amino acids before and after hydrolysis.

Due to the large difference in reaction rate between the trypsin I and trypsin II attacks, the enzymatic degradation of peptide C with this cnzymc was usually carried out at a molar ratio of trypsin to peptide of the order of l/50 to 1 /lOO for 5 to 24 hours at 25’, pH 7.5, in the prcsenct of toluene. Only lysine is released in the trypsin II reaction, indicating that the amino acid must occupy a terminal position in peptide C. Its presence at the NH&erminal position of the peptide was proven by end-group analysis with FDB, while peptide B, resulting from the tryptic attack, was shown to possess glutamic acid as an NHz-terminal residue. When peptide B is converted to pcptide A by heating 30 minutes at loo”, it loses its ability to react with FDB.

Leucine aminopcptidasc, reacting on purified peptidc C, rc- leases simultaneously lysinc and a mixture of glutamine and glu- tamic acid, and large amounts of a tetrapeptide possessing an amino-terminal isoleucyl group. This tetrapeptide is attacked only very slowly by the aminopeptidase and limited amounts of a radioactive tripcptide can be obtained. Amino-terminal group analysis on this tripeptide yields dinitrophenylserine in the ether phase, and valino and arginine in the water phase. P32-labeled srrine is also relcascd in small quantities in the enzymatic reac- tion and was clearly iclrntificd as such by paper electrophorcsis at pH 3.6 and 6.5 and by paper chromatography.

Since peptide C was released from a tryptic attack, and since this enzyme is specific for peptide bonds in \vhich the acyl moiety is furnished by a basic: amino acid (31), it was assumed that ar- ginint would be found at the COOH-terminal position of the

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TABLE III

Suntnzury of stepwise enzymatic degradation and end-group analysis of PWabeled phosphopeptides

1703

Enzymatic attack* -

Tr I

Tr II Sonenzymatic heat

conversion AI’ I

AP II AI’ III Cbp B Cbp A ChTl

daterial attacked

Phosphor- ylase (L

c B

Peptide formed

C

B A

Amino acids in PWabeled peptide produced

Lys . Glu-NHz. Ileu I’-Ser Val. .4rg

Glu-XHn. Ileu P-Ser Val. Arg Lys:. PLC-*Ileu P-Ser . Val Arg None

Ileu P-Serf Val . Arg

P-Ser . Val. Arg P-Ser .

Lys. Glu-SH2 Ileu. P-Ser Val No attack No attack

Lys, Glu, Glu-NH2

All All Arg.

4mino acid released

-

-

NHhzrminal amino acid found by FDB

Lys.

Glu. None

Ileu.

Ser.

Lys.

* The abbreviations used are : Tr! trypsin; AP, aminopeptidase; Cpb A and B, carboxypeptidase A and B; ChTr, chymotrypsin; PIE, pyrrolidone carboxyl

pcptide. If any other amino acid was found in place of arginine, that amino acid would also have to be COOH-terminal on the protein. To verify the former assumption, peptide C was sub- mitted to the action of carboxypeptidase B, an enzyme specific

W? NH2 NHa - CH2

8 O=k---cH2

.*;&>‘* \

l’co.N~... HN-C

,W

H A\co.NH...

for peptides possessing basic amino acids in the COOH-terminal positions (8)) and indeed, arginine was quantitatively released while the rest of the peptide molecule remained unattacked. Carboxypeptidase A was without action on peptide C, as was chymotrypsin. Table III summarizes the above results which establish the structure and amino acid sequence of the phospho- peptidrs A, B, and C.

DISCUSSION

The conversion of phosphorylase b to a was previously shown to result in the transfer of 4 terminal phosphate groups from 4 moles of ATP, to give 1 mole of phosphorylase a (4), in line with the concept that this enzyme is made up of 4 subunits each with a molecular weight of 125,000 (32). The finding that all the phosphate incorporated in the above reaction is attached to the same amino acid sequence indicates that a specific site on the surface of the enzyme must be involved, presumably located on each subunit. The probability that one subunit would contain two structurally identical sequences, while another would con- tain none, would be indeed very low.

Consideration of the structure and reactions of peptide C indi- cates that the site phosphor)-lated by phosphorylase b kinase must be highly basic. The hexnpeptide C, as obtained by tryp- tic attack, contains lysine and arginine, and the positive charges are not neutralized by the presence of an acidic amino acid. In addition, unless this peptide occupies an NHz-terminal position in the protein molecule itself, it must be attached to the rest of the molecule at its amino end to still another basic amino acid in

order to satisfy the specificity requirements of trypsin.7 In view of the selectivity and velocity with which this peptide is liberated by trypsin, it can be assumed that an arginyl-lysine or a lysyl-lysine bond is cleaved. The accumulation of positive charges on this exposed segment of the phosphorylase molecule, together with the particular configuration of the peptide chain itself (the seryl group being set between two nonpolar isoleucyl and valyl residues) must contribute to the highly specific char- acter of the phosphorylase b to a conversion reaction. This is shown by the fact that (a) out of 36 to 46 seryl residues present in a phosphorylase subunit: only one is phosphorylated; (b) of all the low or high molecular weight substrates that have been tested, including several proteins or enzymes (3), none was found to function as phosphate acceptor in the kinase reaction. Fi- nally, no other enzyme than the specific phosphorylase phospha- tase succeeded in hydrolyzing this seryl-bound phosphate from the protein.9

The connection between the phosphorylation of phosphoryl- ase b and its dimerization on the one hand, and change in ac- tivity requirements on the other, is still unknown. With respect to the former phenomenon, no phosphodiester bonds are formed in the phosphorylasc b to a reaction, and the possibility that the four phosphate groups incorporated into the phosphorylase a molecule could act directly in the dimerization process by che- lation of a metal ion could not be demonstrated (4). Apparently, the dimerization of phosphorylase b immediately follows the phos- phorylation of the protein, as if the groups involved in the di- merization were prevented from interacting in the first place. An electrostatic repulsion created by the high concentration of posi- tive charges next to the site of phosphorylation could be respon- sible for maintaining the enzyme in the monomeric form. A par-

7 The assumption that peptide C would occupy a NHz-terminal position in the protein would make it very difficult to explain the 500-fold difference in rate of liberation of peptide B by trypsin from the protein and from peptide C.

8 Calculated from the amino acid composition of phosphorylase determined by Velick and Wicks (33).

9 The details of this study on the effects of various enzymes on phosphorylase a and on the phosphorylated hexapeptide will be reported in a following publicat.ion.

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1704 Phosphopeptide from Phosphorylase a Vol. 234, No. 7

tial neutralization of these positive charges, or a modification of SUMMARy

the spatial configuration of the protein, afforded by the intro- The structure of the site involved in the conversion of phosphor- duction of a negatively charged phosphate group could enable ylase b to phosphorylasc a has been investigated with the use of the dimerization to take place. It is interesting to recall that in P32-labeled phosphorylase a. the activation of trypsinogen (34) or pepsinogcn (35, 36), highly 1. The phosphate introduced in the conversion reaction is

negatively or positively charged pcptides are involved. stable to acids and labile to bases. This behavior excludes a

The change in enzymatic activity as it occurs in the conver- “high energy” type of phosphate linkage but is characteristic

sion of phosphorylase b to a is probably much more directly re- for phosphoproteins in which the phosphate group is linked to an

lated to the phosphorylation of the protein molecule than to its 0-seryl residue.

dimerization. Indeed, in the conversion of inactive liver phos- 2. In a practically instantaneous reaction, trypsin releases all

phorylase to its active form in which a phosphorylation of the the bound phosphate in a soluble organic form whereas over 95 per cent of the protein still precipitates in%richloroacetic acid.

protein also occurs (3$), no dimerization takes place. Further- 3. Purification of the radioactive material by ion exchange more, no difference in sedimentation has been observed between chromatography, paper electrophoresis and chromatography,

lobster muscle phosphorylase b and a (38). followed by stepwise enzymatic degradation and end-group anal-

In addition to its four phosphate groups introduced during the ysis, indicated that the major phosphopeptide obtained had the

phosphorylase b to a conversion, phosphorylase a contains four following sequence :

additional phosphates in the form of pyridoxal 5’-phosphate (10, Lys. Glu-SH2. Ileu Ser-P. Val. Arg.

11). The vitamin Bg derivative was shown to be linked to an e- 4. Two other phosphorylated peptides were obtained which amino group of lysine in the protein (39), but the lysyl residue were shown to result, (a) from a further tryptic attack of the

involved is not the one in pcptide C. Indeed, if the tryptic at- hexapeptide, liberating the NHz-terminal lysine residue and

tack of phosphorylase a is stopped with boiling alcohol or acetone (b) from a nonenzymatic dcamidation and cyclization of this

rather than with trichloroacetic acid, the radioactive peptides pentapeptide to yield a ninhydrin negative, fluorodinitrobenzene

that remain in solution arc free of pyridoxal phosphate which negative material.

remains attached to the denatured protein. Moreover, it has re- 5. The structure of these peptides in relation with the conver-

cently been shown that the lysyl residue to which pyridoxal phos- sion of phosphorylase b to a has been discussed.

phate is bound, is itself linked to a phenylalanyl residue, in a Acknowledgments-We would like to thank Dr. G. H. Dixon and sequence different from that existing in peptide C. Dr. J.-F. Pechere for their valuable advice and discussion.

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KrebsEdmond H. Fischer, Donald J. Graves, Eloise R. Snyder Crittenden and Edwin G.

Reactiona to bStructure of the Site Phosphorylated in the Phosphorylase

1959, 234:1698-1704.J. Biol. Chem. 

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