y-glutamylcysteine* › content › 201 › 2 › 573.full.pdf · from acetone-dried pigeon liver...

SYNTHESIS OF GLUTATHIONE FROM y-GLUTAMYLCYSTEINE*

BY JOHN E. SNOKE, SAM YANARI, AND KONRAD BLOCH

(From the Department of Biochemistry and the Institute of Radiobiology and Biophysics, University of Chicago, Chicago, Illinois)

(Received for publication, October 13, 1952)

Studies on the enzymatic synthesis of glutathione in pigeon liver extracts have demonstrated that y-glutamylcysteine is the intermediary product and that ATP is required for the synthesis of both bonds in the tripeptide (1). The over-all process may therefore be formulated as follows:’

L-Glutamic acid + L-cysteine ATP

---+ L-r-glutamyl-L-cysteine (1)

L-r-Glutamyl-n-cysteine + glycine ATP

--+ GSH (2)

For the purpose of further characterization, the purification of the enzyme system catalyzing Reaction 2 was undertaken. The reasons for studying this step rather than Reaction 1 were twofold. A method for the direct analysis of y-glutamylcysteine is not at present available, while Reaction 2 can be followed by determining the incorporation of C14-glycine into GSH which is readily isolated by carrier technique (2). Secondly, since Reaction 2 yields a true peptide bond, the mechanism involved may be more relevant to the general problem of peptide bond synthesis than the synthesis of y-glutamylcysteine which contains a bond not found in polypeptide chains.

EXPERIMENTAL

Materials-C14-Glycine, y-glutamylcysteine, and cysteinylglycinewere the same preparations used previously (1). L-r-Glutamylglycine was obtained as a gift from Dr. Heinrich Waelsch. GSH was a product of the Schwarz Laboratories, and ATP was purchased as the disodium salt from the Pabst Brewing Company. The non-isotopic amino acids, protamine sulfate, and 3-PGA were commercial preparations. Lyophilized rabbit muscle extract was prepared according to the directions of Ratner and Pappas (3). Potato nucleotide pyrophosphatase (4) was kindly supplied by Dr. Arthur Korn- berg. Mr. Leon Clark prepared aminomethanesulfonic acid (5) and sar-

* Aided by grants-in-aid from the Division of Research Grants and Fellowships of the United States Public Health Service and from the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago.

1 The following abbreviations have been used: GSH, glutathione; ATP, adenosinetriphosphate; ADP, adenosinediphosphate; Tris, tris(hydroxymethyl)aminometh- ane; 3-PGA, 3-phosphoglyceric acid.

573

by guest on June 13, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

574 GLUTATHIONE SYNTHESIS

cosine. The latter was obtained by the hydrolysis of creatine (6) to insure the complete absence of glycine. Pigeon liver extracts were prepared from either acetone-dried pigon liver made in the conventional manner or from acetone-dried pigeon liver kindly supplied by Dr. M. Mitz of Armour and Company.

Methods-The enzymatic synthesis of GSH from y-glutamylcysteine and glycine was carried out in stoppered test-tubes at 37”. The composition of the incubation mixtures is described in Tables I to VI and Figs. 1 to 5. GSH synthesis was followed either by isotopic assay (2), or by measuring the release of inorganic phosphate from ATP as described under “Results.” Inorganic phosphate was determined by the method of Gomori (7), and hydroxamic acid formation was measured according to the procedure of Lipmann and Tuttle (8). The protein concentrations during fractionation were determined by a micro-Kjeldahl method or in the last stage of purification by light absorption at 260 and 280 rnp (9). The energy require- ments of the system were met by adding either ATP alone or by the generation of ATP with the aid of 3-PGA and in the presence of a lyophilized preparation of rabbit muscle extract which contained the appro- priate glycolytic enzymes (3). The amounts of rabbit muscle extract and 3-PGA were chosen to provide optimal synthesis of GSH. The role of the glycolytic process and ATP regeneration in the present system are discussed in the accompanying paper (10).

The purified enzyme, unlike the crude pigeon liver extract (2), does not synthesize GSH when ATP is replaced by ADP. The difference is pre- sumably due to the removal during fractionation of the glycolytic enzymes which generate ATP.

Results

Preparation of Enzyme-Fractionations were carried out either in a cold room set at 0” or in an ice bath. For centrifugations a Servall superspeed angle centrifuge was used. 50 gm. of acetone-dried pigeon liver were ground to a fine powder in a mortar and then stirred for 1 hour in a mixture con- sisting of 500 ml. of 0.9 per cent sodium chloride and 50 ml. of 0.4 M sodium bicarbonate. The supernatant obtained upon centrifugation was dialyzed for 20 hours against 20 liters of distilled water at pH 8.0. The dialyzed extract was brought to pH 5.80 by the addition of 1.0 M acetic acid and stirred for 10 minutes. To the supernatant obtained on centrifugation was added 0.67 volume of ammonium sulfate solution, saturated at O”, and the pH was adjusted to 5.2 with 1.0 M acetic acid. After stirring for 30 minutes, the precipitate was collected by centrifugation, dissolved in a minimum of 0.1 M sodium bicarbonate, and dialyzed against distilled water at pH 8.0 until free of salt. The final step consisted of adjusting the dia-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

J. E. SNOKE, S. YANARI, AND K. BLOCH 575

lyzed solution to pH 4.0 with acetic acid and adding a solution of protamine sulfate (0.1 mg. of protamine sulfate per mg. of protein). The pH was slowly brought to 5.7 by the addition of 1.0 M sodium hydroxide. After stirring for 30 minutes, the precipitate was collected by centrifugation and extracted with 10 ml. of 0.1 M acetate buffer, pH 4.5. The insoluble material was removed by centrifugation, and to the supernatant was added an equal volume of saturated ammonium sulfate. The mixture was stirred for 30 minutes and centrifuged, and the precipitate dissolved in a minimum of 0.1 M sodium bicarbonat,e. It was then dialyzed until free of salt. In Table I are shown the activities and yields obtained in the course of a

TABLE I

Fractionation of Pigeon Liver Enzyme

step

Dialyzed extract.. Acid ppt., pH 5.8.. . (NH,)zSOa ppt.. . Protamine ppt.. .

VOIUIIZ Protein Total activity* Specific activityt

ml. mg. $w ml.

440 31.0 886 0.065 450 18.5 915 0.11

51 26.1 812 0.61 13 10.0 450 3.47

The starting material was 50 gm. of acetone-dried pigeon liver. The activity of the various fractions was determined at pH 8.3 and 37” by incubating the enzyme for 30 minutes in the presence of 0.1 1~ Tris buffer, 0.1 M KCl, 0.01 M MgSOn, 0.015 M

potassium cyanide, 0.002 M -y-glutamylcysteine, 0.03 M C%glycine, 0.001 M ATP, 0.005 M 3-PGA, and 1.0 mg. of rabbit muscle preparation per ml. of reaction mixture. The total volume of the reactionmixture was 1.0 ml. The amount of enzyme was chosen to provide synthesis of GSH at a rate linear with respect to time and proportional to the protein concentration.

* Expressed as micromoles of GSH synthesized per 30 minutes. t Expressed as micromoles of GSH synthesized per 30 minutes per mg. of protein.

typical fractionation. The final fraction which had a specific activity 50 times greater than the original ext’ract was used in all subsequent experiments unless otherwise noted. The enzyme at any stage of the fractionation can be stored in the frozen state at - 18” without loss in activity for at least 3 months.

Effect of Enzyme Concentration-In Fig. 1 are shown the rates of GSH synthesis obtained over an g-fold range of enzyme concentration, while the inset graph demonstrates that synthesis is proportional to the amount of enzyme present. In the experiments shown in Fig. 1 ATP was contin- uously regenerated and glycine was not limiting. Under these conditions the reaction follows zero order kinetics until approximately 60 per cent of the y-glutamylcysteine has been converted to GSH.

E$ect of Substrate Concentration-The initial rate of GSH synthesis was


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


determined at various concentrations of y-glutamylcysteine, glycine, and ATP. The data obtained were plotted according to one of the Line- weaver-Burk equations (11) as is shown in Fig. 2. The K, values cal- culated from the data in Fig. 2 are 2.5 X 1O-4 M, 6.0 X 10-4 M, and 1.1 X 1O-4 M for y-glutamylcysteine, glycine, and ATP, respectively.

.6

0 IO 20 30 40 50 60 Minutes

FIG. 1. Rate of GSH synthesis from r-glutamylcysteine at various enzyme concentrations. The experimental conditions were the same as those given in Table I, except that the volume of the incubation mixture was 7.0 ml. The amount of enzyme in mg. of protein per ml. of reaction mixture is given by 0 0.56, A 0.28, 0 0.14, and A 0.07. At the various time intervals indicated, 1.0 ml. aliquots were withdrawn and the amount of GSH synthesized was determined. In the inset graph the activity is given in micrograms of GSH synthesized per 10 minutes.

E$ect of Cations-The effect of potassium and magnesium ions on GSH synthesis from y-glutamylcysteine and glycine is shown in Fig. 3. There is an absolute requirement for magnesium ions, the concentration being optimal at 0.01 M. Other divalent cations, manganese, cobalt, nickel, and zinc, are not only incapable of replacing magnesium but produce practically complete inhibition of synthesis when tested in the presence of magnesium. In the presence of 0.01 M magnesium, 0.01 M barium and calcium ions produce 45 and 84 per cent inhibition, respectively. Potas- sium ions markedly stimulate synthesis, the optimal concentration being 0.1 M. Other monovalent cations, sodium, ammonium, or lithium, do not


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

J. E. SNOKE, S. YANARI, .IND I<. BLOCH 577

show this effect nor do they counteract the stimulation produced by potassium.

E$ect of pH--The effect of pH on the synthesis of GSH from y-glutamylcysteine and glycine, as measured by the liberation of inorganic phosphate, is shown in Fig. 4. The enzyme is active over a fairly broad pH range with an optimum between pH 8.2 and 8.6.

FIG. 2. Effect of substrate concentration on GSH synthesis. The experimental conditions were the same as those given in Table I, except that the concentrations of glycine A, -,-glutamylcysteine l , and ATP 0 were varied. The rate of synthesis, v, is expressed in micrograms of GSH per 30 minutes, and the concentration of the substrates, a, is in moles per liter. The amount of enzyme in each system was 0.15 mg. of protein per ml.

E$ect of Reducing and Xulfhydryl Reagents-The synthesis of GSH from y-glutamylcysteine is enhanced slightly by the addition of either potassium cyanide or cysteine as shown in Table II. GSH, on the other hand, pro- duces a definite inhibition. It is possible that a SH effect of GSH is ob- scured by product inhibition. Cyanide or cysteine may act either by reducing oxidized sulfhydryl groups on the enzyme or by maintaining the subskate y-glutamylcysteine in the reduced state.2 The interpretation of

2 The disulfide of -y-glutamylcysteine is not convetted to the tripeptide.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


the results obtained with sulfhydryl-blocking reagents shown in Table II is likewise inconclusive. The irreversible alkylating reagents, iodoacetate and iodoacetamide, produce only slight inhibition. This failure to inhibit does not necessarily rule out the participation of sulfhydryl groups of the enzyme protein in the reaction studied (12). The inhibition produced by

400

-3 -2 -I 0

LOG METAL ION CONCENTRATION

FIG. 3. Effect of magnesium and potassium ions on GSH synthesis. The incubation mixture, total volume 1.0 ml., was incubated at 37”, pH 8.3, for 30 minutes and contained 0.1 M Tris buffer, 0.015 M potassium cyanide, 0.002 M r-glutamylcysteine, 0.001 M ATP, and 0.03 M CWglycine. In the experiments in which the potassium concentration was varied (A), the magnesium concentration was 0.01 M. In the experiments in which the magnesium concentration was varied ( l ), the potassium concentration was 0.02 M. The amount of enzyme in the above systems was 0.50 mg. of protein per ml.

p-chloromercuribenzoate was only moderate in spite of a rather high concentration of this sulfhydryl-blocking reagent.

Inorganic Phosphate Release-The fractionation procedure that has been employed results in the removal of ATPase activity. With the purest enzyme fraction obtained, the quantities of inorganic phosphate formed from ATP by ATPase action are less than 5 per cent of those formed as a result of GSH synthesis. Since this blank value is not increased by the addition of either y-glutamylcysteine or glycine alone, it became possible to follow the phosphate liberation from ATP which accompanies peptide synthesis. In Fig. 5 is shown the rate of GSH synthesis as measured by


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


C14-glycine incorporation and the concurrent appearance of inorganic phosphate. For every micromole of GSH formed an equimolar amount of inorganic phosphate is found to be liberated. In view of this equivalence, t,he release of inorganic phosphate in this system can be used as a quantitative measure of peptide bond formation. The rate curve presented in Fig. 5 is not linear, in contrast to those given in Fig. 1. In the experiment of

200 -

I50 -

5 ti a. 100 -

50 -

6.0 I

7.0 I

6.0

PH

I I 9.0 IQ0

i

FIG. 4. Effect of pH on the liberation of inorganic phosphate due to GSH synthesis (upper curve) and hydroxamic acid formation (lower curve). The experimental conditions were the same as those given in Fig. 3, except that the magnesium and potassium concentrations were 0.01 and 0.1 M, respectively, and the buffer wastaried. The buffers used were as follows: 0 0.1 M imidazole, A 0.1 M Tris, 0 0.03 M glycine, W 0.1 M alanine. In the lower curve, the glycine was replaced by 0.40 M hydroxylamine. The amount of enzyme present in the above systems was 0.30 mg. of protein per ml.

Fig. 5, added ATP was the sole energy source and the gradual decline of the rate may be attributed to the inhibition produced by the accumulation of ADP, as discussed in the accompanying paper (10).

The recent finding of Lipmann et al. (13), that pyrophosphate and adenylic acid are formed from ATP in the course of acetate activation, prompted consideration of a similar mechanism for ATP utilization in GSH synthesis. Although the data presented in Fig. 5 make it unlikely that pyrophosphate is a product of GSH synthesis, it was considered necessary to test this point more rigidly because of the presence of inorganic pyrophosphatase activity in the enzyme preparation. That the results in Fig.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

580 GLUTATHIONE SY>:THESIS

5 are not fortuitous due to limiting pyrophosphatase activity is shown in Table III. Fluoride markedly inhibits the hydrolysis of added pyrophosphate but leaves the ratio of orthophosphate liberated to GSH synthesis unchanged. Cysteine was found to stimulate pyrophosphatase activity markedly in this system, but increased the amount of phosphate liberated during GSH synthesis only slightly. In GSH synthesis, therefore, orthophosphate and not pyrophosphate must be the primary split product of ATP.

Effect of Replacing Glycine by Various Acceptors-The liberation of inorganic phosphate which accompanies GSH synthesis can be used as a tool

TABLE II

Effect of Reducing and Sulfhydryl Reagents on GSH Synthesis

Experiment No. Additions Phosphate liberated

1 None 2 1.5 X 1OP M cysteine 3 1.5 X 1O-2 “ KCN 4 1.5 x 10-Z “ GSH 5 0.5 x 10-z “ “

6 1.0 X IOP “ iodoacetate 7 1 .O X 10-s “ iodoacetamide 8 1.0 X 10-s “ p-chloromercuribenaoate

IrM

0.57 0.67 0.69 0.39 0.48 0.59 0.61 0.41

The experimental conditions were the same as those given in Fig. 3, except that the magnesium and potassium concentrations were 0.01 and 0.1 M, respectively, and potassium cyanide was omitted in Experiments 1, 2, 4, and 5. In experiments 6 to

8 the sulfhydryl reagents at the concentrations given were preincubated with the enzyme for 30 minutes at 37”. During enzymatic synthesis, the concentration of the sulfhydryl reagents was 4 X 1OP M. The amount of enzyme present in the incubation mixtures was 0.30 mg. of protein per ml.

to determine whether the coupling of -y-glutamylcysteine occurs with acceptors other than glycine. In Table IV are given the amounts of inorganic phosphate liberated when glycine was replaced by a variety of compounds. At concentrations which are optimal for glycine, none of the compounds tested in combination with y-glutamylcysteine caused the release of inorganic phosphate from ATP. A high degree of specificity of the enzyme for glycine is therefore evident. Phosphate release does, however, occur with hydroxylamine, provided the concentration of the amine is very high. For example, 0.4 M hydroxylamine must be used to achieve the same phosphate release that is obtained with 5 X 10M4 M glycine. This release of inorganic phosphate is a quantitative measure of hydroxamic acid formation, since t,he two are formed in equivalent, amounts (Table V) . This reaction shows


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


the same magnesium requirement and pH dependence (Fig. 4) as the synthesis of GSH. It is assumed that the derivative which forms is r-glu- tamylcysteinylhydroxamic acid.3

Miscellaneous Experiments-Various attempts have been made to ob- tain evidence for an intermediate in the conversion of y-glutamylcysteine to GSH. As pointed out previously, the incubation of enzyme and ATP with either y-glutamylcysteine or glycine alone fails to liberate inorganic

0.6 ,

0.5

w

4

E 0.4 f

z i= 0.3 2

; 0.2

x

0.1

0 IO 20 30 40 50 60

MINUTES

FIG. 5. Rate of GSH synthesis from y-glutamylcysteine and concurrent liberation of inorganic phosphate. The incubation mixture was the same as that given in Fig. 3, except the total volume was increased to 15.0 ml. and the magnesium and potassium concentrations were 0.01 and 0.1 M, respectively. At the various time intervals indicated 1.0 ml. aliquots were withdrawn and assayed for either GSH synthesis ( l ) or inorganic phosphate (A). The amount of enzyme in the incubation

mixture was 0.15 mg. of protein per ml.

phosphate. To test whether a phosphorylated intermediate of one of the reactants might accumulate, enzyme and ATP were incubated with either y-glutamylcysteine or glycine. ATP was then destroyed by the addition of highly purified nucleotide pyrophosphatase (4). Subsequently the system was made complete by the addition of -r-glutamylcysteine or glycine and the incubation was continued. As is seen from the data in Table VI, no evidence was obtained for the formation of an intermediate which could form GSH under these conditions. Attempts to accumulate an inter-

3 A preliminary investigation has revealed that the hydroxamic acid contains a sulfhydryl group, travels on paper chromatograms at approximately the same rate as y-glutamylcysteine, and yields glutamic acid on hydrolysis.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


mediate which could react non-enzymatically with hydroxylamine were likewise negative. In this case, glutamylcysteine, ATP, and enzyme were incubated and the mixture was deproteinized by boiling. Neither the boiling nor the subsequent addition of hydroxylamine to the deproteinized mixture led to the liberation of inorganic phosphate. The negative out-

TABLE III

E$ect of Fluoride and Cysteine on GSH Synthesis and Pyrophosphatase Activity

Experiment No. Additions

0.5 X lo-’ M fluoride 1.0 x 10-d “ “ 3.3 x 10-4 “ “

3.0 X 10m3 “ cysteine

I GSH synthesis

w 0.36 0.36 0.33 0.29 0.46

Pyrophosphatase activity, px

orthophosphate

0.80 0.57 0.45 0.25 2.0

The experimental conditions for GSH synthesis were the same as those given in Table II, except for the additions which are given above. Pyrophosphatase activity was measured under the same conditions, except that ATP and y-glutamylcysteine were replaced by 1.5 PM of pyrophosphate. The amount of enzyme present in the above systems was 0.15 mg. of protein per ml.

TABLE IV

Replacement of Glycine by Various Acceptors

Acceptor

Glycine. Hydroxylamine.

‘I 11

Semicarbazide Aminomethanesulfonic acid

“ “

Glycolic acid. . nn-Alanine Methylamine. Sarcosine.. Ammonia. Ethanolamine.

-

-

Concentration Phosphate liberated

&I sx

0.03 0.74 0.04 0.08 0.10 0.17 0.40 0.45 0.40 0.14 0.04 0.06 0.40 0.11 0.40 0.08 0.40 0.05 0.40 0.04 0.40 0 0.40 0 0.40 0

-

The experimental conditions were the same as those given in Table II, except that the glycine was replaced by the various compounds shown. Unless otherwise indicated, the replacement of glycine by the other compounds failed to cause the liberation of phosphate when tested at the concentration of 0.04 M. The amount of enzyme in the above systems was 0.30 mg. of protein per ml.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


come of these experiments appears to indicate that in the conversion of the dipeptide to GSH intermediates do not accumulate in detectable quantities. This conclusion will however be valid only if, as has been assumed,

Time

min.

20 40 60

-

TABLE V

Formation of Hydroxamic Acid

Hydroxamic acid Phosphate liberated

p‘w per ml. p.v per ml.

0.25 0.21 0.33 0.35 0.45 0.48

The experimental conditions were the same as those given in Table II, except that the glycine was replaced by 0.4 M hydroxylamine and the total volume of the incubation mixture was 10.0 ml. At the time intervals indicated, 1.0 ml. aliquots were withdrawn for inorganic phosphate determination and 2.0 ml. aliquots for hydroxamic acid determination. Succinic anhydride was used as a standard in the hydroxamic acid assay. The amount of enzyme in the incubation mixture was 0.30 mg. of protein per ml.

TABLE VI

Attempt to Demonstrate an Intermediate in GSH Synthesis

step 1

Additions GSH

I step 2 I synthesized

step 3

1

2 3

4

Glutamylcysteine Nucleotide pyro- + ATP phosphatase

Cl4-Glycine + ATP “ “ ATP “ “

CWGlycine

Glutamylcysteine “

+ C14-glycine Glutamylcysteine

+ CWglycine

Y

0

0 0

226

The incubation mixtures in Step 1 were the same as those given in Table II, except for the omission of y-glutamylcysteine in Experiments 2 to 4 and the omission of glycine in Experiments 1, 3, and 4. All three steps were carried out for 30 minutes at 37”, pH 8.3. The amount of nucleotide pyrophosphatase was sufficient to hydrolyze completely the ATP present. The amount of enzyme in the above mixtures was 0.30 mg. of protein per ml.

the hypothetical intermediate were resistant to the action of the nucleotide pyrophosphatase and if the reaction with hydroxylamine were non-enzymatic.

If GSH formation involves a series of recersible reactions which lead to either a glutamylcysteinyl-enzyme or glycyl-enzyme complex with the simultaneous release of inorganic phosphate, Ps2 from P32-orthophosphate


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


should be incorporated into ATP in analogy with the phosphate exchange observed with glucose-l-phosphate (14) and acetyl phosphate (15). The results from an experiment designed t,o test this possibility were negative. Enzyme, ATP, and P32-orthophosphate were incubated with either r-glutamylcysteine or glycine. After partition of inorganic phosphate and ATP (16), no significant incorporation of P32 in the ATP was found to occur.

According to Waelsch (17), GSH can be synthesized from y-glutamylglycine and cysteinylglycine by a transfer reaction in the absence of an external energy source. It was also suggested (17) that the interaction of y-glutamylcysteine and r-glutamylglycine might lead to the same result. In order to ascertain whether GSH is formed by either of the pro- posed mechanisms, these two dipeptide pairs were added to the initial pigeon liver extract which catalyzes both the formation of y-glutamylcysteine and its condensation with glycine (1). The combination of y-glutamylglycine + cysteinylglycine or of y-glut’amylcysteine + y-glutamylglycine failed to give rise to GSH as measured by the glyoxalase method (18). It may be concluded, therefore, that the suggested transfer reactions are not responsible for GSH formation in the present system.

Incubation of the enzyme with GSH itself does not lead to the formation of free amino acids as determined by paper chromatography. This was tested over a pH range from 4 to 8 and in the presence of various divalent ions. The earlier conclusion that the synthesis and hydrolysis of GSH are catalyzed by separate enzymes (2) is therefore confirmed. Incubation of the enzyme with GSH, C14-glycine, and ATP both in the presence and absence of inorganic phosphate does not result in the incorporation of labeled glycine into the tripeptide, and hence the enzyme must be considered incapable of catalyzing an exchange of the glycine moiety of GSH.

The fractionation procedure employed has given no evidence that more than a single enzyme participates in the synthesis of GSH from r-glutamylcysteine. Recombination of the various fractions obtained during the enzyme purification, as described in Table I, yielded activities which were equal to the sum of the individual activities. Similar results were obtained on recombination of various fractions obtained by ethanol pre- cipitation.4 Present evidence, furthermore, speaks against the participation of a dissociable cofactor. Dialysis for 48 hours at neutral pH, or for 7 hours at pH 3.8 or 9.8, as well as the use of Norit or Dowex to adsorb possible cofactors, failed to have any effect on the activity of the enzyme.

DISCUSSION

The equivalence between the amounts of tripeptide formed and inorganic phosphate released and the inability of adenosinediphosphate to

4 Unpublished experiments.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


replace ATP indicate that the energy required for the synthesis of GSH from y-glutamylcysteine is obtained through the splitting of only the terminal phosphate of ATP. None of the experiments carried out so far have revealed further details as to the mechanism by which ATP is utilized. The formation of an active intermediate is, however, suggested by the reaction of y-glutamylcysteine with hydroxylamine at high concentrations of this amine. Compounds which are structurally more similar to glycine than is hydroxylamine are, on the other hand, incapable of acting as acceptors, even at high substrate concentrations. These results suggest that the product of the enzymatic interaction between y-glu- tamylcyateine and ATP reacts either enzymatically with glycine to form GSH or non-enzymatically with hydroxylamine.

It is conceivable that the active intermediate fails to accumulate because it is bound to the enzyme through a chemical bond. In this case the extent of the reaction in the absence of acceptor should be limited by the number of active sites of the enzyme molecules.

It is of interest to compare the present system to others which catalyze related reactions. Although GSH synthesis is formally similar to acetyla- tion reactions in that both systems involve an activation of carboxyl groups, the mechanism by which ATP is utilized cannot be the same. The activation of acetate involves the pyrophosphorylation of coenzyme A and the subsequent formation of acetyl coenzyme A with a release of inorganic pyrophosphate and adenylic acid (13). In GSH synthesis substrate activation appears to occur in the absence of dissociable cofactors and a pyrophosphorylation step can be ruled out.

A comparison of GSH synthesis and glutamine formation reveals strik- ing similarities. The synthesis of glutamine, like the formation of GSH from r-glutamylcysteine, involves the splitting of ATP to orthophosphate and ADP (19, 20). The reaction appears to be catalyzed by a single enzyme and proceeds in the absence of dissociable cofactors (19). The two systems differ, however, in one important respect. The glutamine- synthesizing enzyme has also an exchanging function (19)) which is not found in the enzyme which catalyzes the condensation of r-glutamylcysteine and glycine.

Because of the non-availability of other suitable systems, GSH synthesis has so far remained the only model for the study of peptide formation de novo from free amino acids. It is not known whether the mechanism of GSH synthesis, as studied here, is representative of peptide synthesis in general. It is of interest in this connection to consider the properties of the various types of enzymes which catalyze the formation of peptide or peptidic bonds. Proteolytic enzymes, in addition to hy- drolyzing their substrates, can also catalyze the replacement of the imino component by another (21) and therefore give rise to new peptides. This


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

586 ~LUTATHIONE SYNTHESIS

process does not require an external source of energy nor does it result in the net formation of peptide bonds. That a transferring function need not be associated with ability to hydrolyze is illustrated by the glutamine system. Glutamine synthesis from glutamic acid and ammonia requires ATP and is catalyzed by an enzyme which can effect an amide exchange without hydrolysis of the amide bond (19). Finally, the enzyme responsible for GSH synthesis from y-glutamylcyst,eine and glycine has neither a hydrolytic nor a transferring action and appears to catalyze a synthetic reaction only.

SUMMARY

The enzyme which catalyzes the condensation of y-glutamylcysteine and glycine has been purified approximately 50-fold from extracts of acetone-dried pigeon liver. The synthesis of glutathione has an absolute requirement for magnesium ions and is stimulated markedly by potassium ions. The pH optimum and effect of enzyme and substrate concentrations have been studied. The energy necessary for the synthesis of glutathione is obtained through the splitting of the terminal phosphate of adenosinetriphosphate. Replacement of glycine by hydroxylamine at high concentrations results in the formation of a hydroxamic acid derivative of y-glutamylcysteine. Experiments bearing on the mechanism of glutathione synthesis are described.

BIBLIOGRAPHY

1. Snoke, J. E., and Bloch, K., J. Biol. Chem., 199,407 (1952). 2. Johnston, R. B., and Bloch, K., J. Biol. Chem., 188, 221 (1951). 3. Ratner, S., and Pappas, A., J. Biol. Chem., 179,1183 (1949). 4. Kornberg, A., and Pricer, W. E., Jr., J. Biol. Chem., 182, 763 (1950). 5. Reinking, K., Dehnel, E., and Labhardt, H., Ber. them. Ges., 38, 1077 (1905). 6. Bloch, K., Schoenheimer, R., and Rittenberg, D., J. Biol. Chem., 138,155 (1941). 7. Gomori, G., J. Lab. and CZin. Med., 27,955 (1940). 8. Lipmann, F., and Tuttle, L. C., J. BioZ. Chem., 169,21 (1945). 9. Kalckar, H. M., J. BioZ. Chem., 187, 461 (1947).

10. Yanari, S., Snoke, J. E., and Bloch, K., J. BioZ. Chem., 201, 561 (1953). 11. Lineweaver, H., and Burk, D., J. Am. Chem. Sot., 66,658 (1934). 12. Barron, E. S. G., Advances in Enzymol., 11, 238 (1951). 13. Lipmann, F., Jones, M. E., Black, S., and Flynn, R. M., J. Am. Chem. Sot., 74,

2384 (1952). 14. Doudoroff, M., Barker, H. A., and Hassid, W. Z., J. BioZ. Chem., 188, 725 (1947). 15. Stadtman, E. R., and Barker, H. A., J. BioZ. Chem., 184, 769 (1950). 16. Zetterstrom, L. E., and Lindberg, O., Acta them. Stand., 4, 942 (1950). 17. Waelsch, H., Advances in Enzymol., 13,237 (1952). 18. Woodward, G. E., J. BioZ. Chem., 109, I (1935). 19. Elliott, W. H., Federation Proc., 11, 207 (1952). 20. Speck, J. F., J. BioZ. Chem., 179, 1405 (1949). 21. Fruton, J. S., Johnston, R. B., and Fried, M., J. BioZ. Chem., 190, 39 (1951).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

John E. Snoke, Sam Yanari and Konrad Bloch-GLUTAMYLCYSTEINEγ

SYNTHESIS OF GLUTATHIONE FROM

1953, 201:573-586.J. Biol. Chem.

http://www.jbc.org/content/201/2/573.citation

Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

alerts to choose from all of JBC's e-mailClick here

tml#ref-list-1

http://www.jbc.org/content/201/2/573.citation.full.haccessed free atThis article cites 0 references, 0 of which can be


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/content/201/2/573.citation

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;201/2/573&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/201/2/573.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=201/2/573&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/201/2/573.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/201/2/573.citation.full.html#ref-list-1

http://www.jbc.org/content/201/2/573.citation.full.html#ref-list-1

http://www.jbc.org/

y-glutamylcysteine* › content › 201 › 2 › 573.full.pdf · from acetone-dried pigeon liver...

Documents