STUDIES ON THE ENZYMIC REDUCTION OF AMINO ACIDS

III. PHOSPHATE ESTERIFICATION COUPLED WITH GLYCINE REDUCTION

BY THRESSA C. STADTMAN, PATRICIA ELLIOTT, AND LYDIA TIEMANN

(From the Enzyme Section, National Heart Institute, National Institutes of Health, Bethesda, Maryland)

(Received for publication, October 23, 1957)

Glycine can be employed by a number of anaerobic bacteria as the elec- tron acceptor in a coupled oxidation-reduction reaction between pairs of amino acids (a Stickland reaction). This process (Reaction 1) involves a reductive deamination of glycine to form acetic acid and ammonia (1).

NH&H&OOH + 2H --f CHsCOOH + NH, (1)

Until recently, little information was available concerning the mechanism of glycine reduction in such a fermentation since earlier attempts to study it in extracts had met with failure (2). It became possible to examine this reaction in some detail in enzyme preparations derived from Cloa- tridium sticklandii (3) when it was discovered that dimercaptans such as DMP’ would function as electron donors (4). The reduction of glycine in this system has proved to be an unexpectedly complex process which results in the formation of ATP (5). The partial purification and some of the properties of this glycine reductase system of C. sticklandii are de- scribed in the present communication.

Materials

Acetyl kinase was prepared from Escherichia wli according to the pro- cedure of Rose et al. (6). Purified phosphotransacetylase was obtained from E. R. Stadtman. Reduced lipoic acid and redistilled BAL were gifts from I. C. Gun&us and H. Tabor, respectively. DMP was synthe- sized according to the method of Stocken (7). Glycine-l-C14 was purchased from the Volk Radiochemical Company, glycine 2-C” from the Nuclear

1 The following abbreviations are employed: BAL, 2,3-dimercaptopropanol; DMP, 1,3-dimercaptopropanol; CoA, coenzyme A; THFA, tetrahydrofolic acid; DPNH, reduced diphosphopyridine nucleotide; DPN, diphoaphopyridine nucleotide; FMN, flavin mononucleotide; FAD, flavine adenine dinucleotide; AMP, adenylic acid; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Tris, tris(hydroxy- methyl)aminomethane; EDTA, ethylenediaminetetraacetate (Versene); TPTA, tri- phenyltetrazolium chloride.

961

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962 ENZYMIC REDUCTION OF AMINO ACIDS. III

Instrument and Chemical Corporation of Chicago, and pyridoxal phosphate from the California Foundation for Biochemical Research, Los Angeles. The various mono- and dinucleotides used were commercial preparations, most of which were obtained from the Pabst Laboratories. Phosphor- amidate and adenosine 5’-phosphoramidate were gifts from Thomas Rosenberg (Gentofte, Denmark) and H. G. Khorana, respectively.

Chemical Methods

Suitable aliquots of reaction mixtures after deproteinization with per- chloric acid (final concentration 3 per cent v/v) were assayed for glycine by the ninhydrin procedure of Cocking and Yemm (8), ammonia by direct, nesslerization, mercaptan by an adaptation of Boyer’s p-chloromercuri- phenylsulfonate procedure for SH- groups (9), and orthophosphate and acid-labile phosphate by the Fiske-Subbarow method (10). The per- chloric acid filtrates were adjusted to pH 6 with KOH and the insoluble potassium perchlorate was removed before chromatographic analysis of re- action mixtures for P@-labeled ATP formed from P32-labeled orthophos- phate (11).

Orthophosphate was estimated in the presence of phosphoramidate and mercaptan by an adaptation of the Lowry-Lopez method (12).

Acetate-Cl4 was estimated in neutralized steam distillates or in neutral- ized aliquots of supernatant solutions freed from residual glycine-Cl4 by treatment with Dowex 50-H+ resin at pH 1 to 2. Identity of the radio- active product was established by Duclaux distillations. Acet.ate-04 iso- lated by distillation procedures was degraded according to the Schmidt reaction (13). Radioactivity measurements were made on the carboxyl carbon recovered as BaCOa and the methylene carbon recovered as meth- ylamine picrate.

Acetate was also estimated as acethydroxamate after incubation wit,h purified acetyl kinase and ATP (6). The high concentration of hydroxyl- amine used as trapping agent completely inhibits the further conversion of glycine to acetate by the glycine reductase enzyme system. Hence, it is unnecessary to inactivate by heat or deproteinize the samples before addition of the acetyl kinase assay components.

The SH- concentration of various mercaptan solutions, except eysteine, was determined by iodine titration just before use.

Protein was estimated by the biuret method (standardized against crys- talline bovine serum albumin) or by ultraviolet absorption methods.’

Incubation Con&ions-Reaction mixtures which contained components indicated under “Experimental” were incubated at 31’ in 10 X 75 mm. stoppered test tubes in an atmosphere of helium or hydrogen. With most preparations, acetate formation was linear with time for incubation

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T. C. STADTUAN, P. ELLIOTT, AFD L. TIEMANN 963

periods up to 2 hours duration. If phosphate esterification also was to be measured, the reactions were terminated after 90 minutes since the activity of various phosphatases became more apparent with continued incubation. Usually, enzyme preparations were employed at 3 to 6 mg. of protein per 0.5 ml. of reaction mixture. Initially, for every preparation, the range of linear response to total protein concentration was determined.

Enzyme Preparations

Sonic extracts of freshly harvested cells of C. sticklandii were prepared as described previously (14). Crude extracts were adjusted to pH 8.1 with Tris and diluted to contain not more than 15 to 18 mg. of protein per ml. 2 per cent protamine sulfate solution was added until the 280:260 ratio of the supernatant fluid was 0.85 to 0.95. For most extracts about 90 mg. of protamine sulfate per gm. of protein were required. The super- natant fluid obtained by centrifugation contained the enzyme activities and the precipitate was discarded.

Precipitation with Ammonium Sulfate--Solid (NH&SOI was added to the protamine supernatant fluid to 0.3 saturation (21 gm. for each 100 ml. of extract) and the small precipitate discarded. The supernatant solution was adjusted to 0.6 saturation with (KH&SO+ centrifuged, and the pre- cipitate (Fraction A) dissolved in 0.05 M Tris buffer, pH 8.7. Fraction A contains about 60 per cent of the protein of the protamine supernatant fluid and most of the glycine reductase activity. Refractionation of this material with solid ammonium sulfate yielded Fraction B (protein precip- itated by addition of 24.5 gm. of (NH&S04 per 100 ml. of enzyme solution), Fraction C (protein precipitated by further addition of 10.5 gm. of (NH&S04 per 100 ml. to the supernatant fluid from Fraction B), and Fraction D (protein precipitated by addition of 7 gm. of (NH&SO4 per 100 ml. to the supernatant fluid from Fraction C). These precipitates were dissolved in 0.05 M Tris, pH 8.7, containing 1O-3 M EDTA.

Calcium Phosphate Gel Treatment -Calcium phosphategel (15) was added to Fraction B (diluted with 0.05 M Tris buffer to a protein concentration of 25 to 35 mg. per ml.) until a gel to protein ratio of 0.92 was attained. The gel precipit,ate was washed once with a volume of cold distilled water equal to that of the original protein-gel suspension and then eluted with a similar volume of 0.1 M potassium phosphate buffer, pH 7.6. The en- zyme in the buffer solution was precipitated by the addition of 28.7 gm. of (NH&SO4 per 100 ml. of solution, collected by centrifugation, and redissolved as above. Any insoluble denatured protein present was re- moved by centrifugation at 104,000 X g for 1 hour. This preparation is designated as Fraction E.

Precipitation at Low pH-Fraction F was prepared either from Fraction

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964 ENZYMIC REDUCTION OF AMINO ACIDS. III

B or Fraction C as follows: The enzyme solution (diluted with water to a protein concentration of not more than 25 mg. per ml.) was usually about pH 8.1 to 8.2, and contained Tris buffer (about 0.02 M) and residual ammonium sulfate. Cold formic acid (0.1 M) was carefully added to such a solution until the pH was 4.9 to 5.0. The resulting precipitate was col- lected by centrifugation and dissolved in Tris buffer as before. Denatured insoluble protein was removed by centrifugation at 104,000 X g for 1 hour and discarded. The above fractionation procedures were all carried out at O-2”. Other methods tried, including solvent precipitation (- 15”) and differential heat inactivation, did not result in significant purification. The fractionation procedures outlined above, although they resulted in only 2- to 5-fold increase in specific activity of the over-all glycine reductase system, were finally adopted because they resulted in elimination, to a large extent, of such undesirable activities as acetyl kinase and mercaptan- activated phosphatases that destroy AMP and ATP. The final steps which yielded Fractions E and F did serve to separate the system into two protein components. Dialysis procedures were not generally feasible since considerable inactivation resulted. Hence, protein fractions obtained by precipitation with ammonium sulfate were assayed without removal of the residual ammonium sulfate.

EXPERIMENTAL

Phosphate (or Arsenate) and Nmdeotide Depend.e&es-In the presence of DMP, which can serve as the electron donor, crude extracts of C. stick- Landis catalyze the reduction of glycine to acetate as described by Reaction 1. However, upon fractionation with ammonium sulfate, a protein frac- tion is obtained (Fraction B) which no longer catalyzes glycine reduction unless orthophosphate and an adenine nucleotide are also added (Table I). As shown in Table I, arsenate will replace both orthophosphate and the adenine nucleotide. The nucleotide requirement is satisfied by either AMP, ADP, or ATP. This lack of specificity probably is due to the fact that the system contains an active adenylate kinase that has not been removed by any of the purification steps employed. Occasionally, prep- arations are obtained that exhibit greater activity with ADP than with AMP. For routine amays, particularly when the extent of phosphate esterification is to be measured, AMP plus a catalytic level of ADP is used.

NucKsotides of inosine, guanidine, uridine, and cytidine do not replace adenine nucleotides or supplement them when phosphate is utilized. Also, they exhibit no effect with arsenate. This is true, as well, for enzyme preparations subjected to dialysis, treatment with Dowex l-Cl, or passage over acid-washed Norit A columns (16).

Sttichiometry-Data obtained from chemical balance studies (Table II)

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T. C. STADTMAN, P. ELLIOTT, AND L. TIEMANN

TABLID I

965

E$ect of Orthophosphute (PO,), Arsenate (AsO,), and AMP on Conversion of Glycine to Acetate

Additions Acetate-Z-Cl’ formed

W?&?lcS rmolu

None* 0.12 PO,, 5; PO,, 10 0.43, 0.54

“ 5 + AMP, 5; PO,, 10 + AMP, 5 1.48, 1.40 AsO,, 5; AsO,, 10 1.29, 1.42

“ 5+AMP,5 1.29 ‘1 5 + PO4,5 1.42

None* 0.02 PO4,5 0.20 AMP,5 0.52 PO4, 5 + AMP, 5 1.09

* The enzyme preparation (Fraction B, not dialyzed) contained 0.88 mole of orthophosphate per 3.6 mg. of protein employed. In addition to the components indicated, each sample contained Tris buffer (pH 8.7) 20 wales, MgClz 3 pmoles, DPN 0.1 pmole, pyridoxal phosphate 0.003 pmole, DMP 9 pmoles, 0.2 ~c. of glycine- 2-V 10 pmoles, and 3.6 mg. of protein (Fraction B) in a final volume of 0.5 ml. In- cubations were carried out anaerobically at 31” for 90 minutes.

TABLE II ATP Synthesis during Reduction of Qlycine to Acetate and Ammonia

NITI formed

Experi- PO4 PIO min. malt No.

Glycine decomposed uptake formed* iti%2

)mdes wnoles pmols pmoles pmoles

1 1.01 1.07 0.98 1.01 1.28t 2 2.521 1.25 0.89 1.21

Reaction mixture components and conditions are the same as those described in Table I except that each sample also contained AMP, 5 rmoles, and KzHPOd, 5 pmoles .

* Amount of phosphate released when heated for 10 minutes at 100” in 1 N acid. t The enzyme preparation used to measure NH8 formation had been precipitated

with saturated NasS04 and redissolved in buffer to lower its (NHJzSC4 concentra- tion.

$ The amount of DMP oxidized was measured in incubation mixtures reduced to one-half the usual volume; all components were added in proportionally smaller amounts except for enzyme and glycine.

show that a more precise picture of the reductive deamination of glycine is that given in Equation 2

CH2(NH,)COOH + R(SH)z + PO4* + ADP + CH&OOH + NH, + R-SS + ATP (2)

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966 ENZYMIC REDUCTION OF AMINO ACIDS. III

Thus for each mole of glycine converted to a mole each of acetate and ammonia, 2 equivalents of SH- are oxidized and there is the concomitant esterification of 1 mole of orthophosphate to form ATP. The ATP formed was identified enzymatically with use of yeast hexokinase and glucoseS- phosphate dehydrogenase (17) as well as chromatographically (11). In experiments carried out with P32-labeled orthophosphate, no labeled ATP was detected in samples from which either glycine or DMP was omitted.

Other Cofactor Repirements-After it was discovered that the reduction of glycine to acetate and ammonia is coupled with the synthesis of ATP, the system was reexamined to determine whether Mg*, DPN, and pyr- idoxal phosphate (4) are also required. Whereas most of the glycine reductase preparations are not influenced by additions of these cofactors, the activity of some preparations is diminished 30 to 50 per cent by omis- sion of either Mg++ (3 to 10 pmoles per 0.5 ml.) or DPN (0.1 to 0.2 pmole per 0.5 ml.). It is concluded that these substances are probably necessary components of the enzyme system but often are already present in amounts sufficient to saturate the enzymes in question. The instability of the glycine reductase system to extensive dialysis has rendered systematic investigation of this aspect of the problem difficult. The status of pyri- doxal phosphate as a coenzyme in the system is still very questionable and only occasionally is a slight stimulation (20 to 30 per cent) due to its ad- dition seen. Complete inhibition of glycine reduction to acetate by NHzOH (1OW M) may, however, be related to a pyridoxal phosphate re- quiremen t .

Degradation of Acetate-P4 Formed from Glycine-C’4-To determine whether or not the carbon skeleton of glycine is maintained intact during its reductive deamination in the C. sticklandii system, acetate samples derived from glycine-2-Cl4 and from glycine-l-Cl4 were isolated, diluted with unlabeled acetate, and degraded chemically.

A sample of acetate from glycine-2-C14, diluted to 26 c.p.m. per pmole, contained 25 c.p.m. per patom of methylene carbon; there was no isotope in the carboxyl carbon. Similarly, acetate formed enzymatically from glycine-l-Cl4 and diluted to 20.6 c.p.m. per pmole, was labeled in the car- boxy1 position to the extent of 19.3 c.p.m. per patom of C and was unlabeled in the methylene position. Thus, no mixing of carbons occurs during this conversion.

Substrate Spec$i&ty-A number of amino compounds, glycine deriv- atives, and other 2-carbon compounds were surveyed for their ability to replace glycine in the glycine reductase system as measured by a concom- itant esterification of orthophosphate. None of the following compounds was active in this respect: glycine anhydride (diketopiperazine), glycine methyl ester, glycylglycine, sarcosine, hydantoin, glycolic acid, glyoxylic

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T. C. BTADTMAN, P. ELLIOTT, AND L. TIEMANN 967

acid, ethanol, acetaldehyde, glycolaldehyde, @-alanine, -y-aminobutyric acid, ethanolamine, serine, proline, valine, citrulline, and lysine. Glycolic acid was also t,ested in the system by the isotope dilution technique. It did not enter the metabolic pathway of glycine to acetate since it neither diluted the radioactive acetate formed from radioactive glycine nor became labeled itself. Thus free glycolic acid is not an intermediate in the con- version of glycine to acet,at,e.

Nature and Spe&$city of Electron Donor System-Dimercaptans are the only electron donors that have been found to function in the isolated gly- tine reductase system of C. sticklandii (Table III). The monomercaptans examined were without activity either alone or when used to supplement suboptimal amounts of DMP. Reduced lipoic acid was inhibitory at levels greater than 0.02 M SH- (final concentration), possibly because the DL

mixture was employed. BAL is much less effective as a reducing agent than is DMP. The same relative activities of these compounds have been observed in the purified n-proline reductase system of this organism (14), whereas a DPN-linked mercaptan dehydrogenase2 (18, l9), also present in Fraction B, displays quite different relative activities with these sub- strates (last column, Table III). For example, t,he DPN-linked enzyme is considerably more active on BAL than on DMP and also uses several monomercaptans to an appreciable extent. Such comparative data pro- vide additional evidence against the mediation of a DPN-linked mercaptan dehydrogenase in the transfer of electrons from mercaptan to amino acid acceptor in the amino acid reduet.ase systems of this organism (20).

The effect of DMP concentration on glycine reduction is shown in Table IV. A final concentration of about 0.015 M dimercaptan (0.03 M SH-) is necessary to saturate the system. This is about half that required for the n-proline reductase system (14).

Characteristics of Fractionated Glycine Red&me System-Separation of the glycine reductase system into two protein components was achieved by treatment of t,he enzyme with calcium phosphate gel (Fraction E) and by acidification (Fraction F). As seen in Table V, neither of these prep- arations, alone, had appreciable ability to cat,alyze the conversion of glycine to acetate, but together t,hey exhibited good activity. Preserved in these preparations was the associat,ed phosphorylation system. In Experiment

2 The enzyme preparation used contains, in addition, a soluble FMN-linked di- aphorase that catalyzes the transfer of electrons from DPNH to TPTA. This di- aphorase is also activated to a lesser degree by riboflavin or folic acid but not at all by FAD. It is not sedimented by centrifugation at 104,000 X g for 3% hours. The addition of a catalytic level of phenazine methosulfate increased the rate of trans- fer of electrons from DPNH to TPTA. Under these conditions, the diaphorase activity, as measured independently in a system wherein DPNH was generated by ethanol and ethanol dehydrogenase, is not rate-limiting.

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TABLE III Relative Activities of Various Reducing Agents As Electron Donors for Glycine

Reduction* and for DPN Reductiont

Reducing agent

DMPS. ................................. DL-6,8-Dimercaptooctanoate. ........... BAL ................................... SHCH&H*OH ......................... SHCH&OONa ........................ Cysteine ................................ H&Y. ................................... Glutathione ........................... THFA ................................. Hz ..................................... None ...................................

Electron acceptor system

Glycine DPN

100 100 60 170 25 305 0 30 0 13

Not determined 70 0 Not determined

Not determined 16 0 Not determined 0 0 0 0

* As measured by the conversion of glycine to acetate; for experimental condi- tions, see Table I. Fraction B was used as the enzyme source and each mercaptan was tested at two or more concentrations in the range of 10 to 30 peq. of SH- per 0.5 ml. (0.02 to 0.06 M SH-).

t DPN-linked oxidation of the various mercaptans was measured in an assay in which TPTA was employed as the ultimate electron acceptor. The mercaptan de- hydrogenase assay system contained the following components: dimethyl glutarate buffer (pH 7.4), 20 pmoles; EDTA, 2.5 pmoles; MgCl,, 5 pmoles; TPTA, 2.8 rmoles; DPN, 0.2 pmole; FMN, 0.065 pmole; phenazine methosulfate, 0.2 y; mercaptan, 0.5 to 2.0 peq.; enzyme, 0.8 mg. in a final volume of 0.5 ml. A control sample containing heated enzyme, but otherwise identical, was prepared for each sample. Incubations were carried out anaerobically in 10 X 75 mm. test tubes at 31” for 30 minutes. Con- ditions were chosen such that the reaction was linear both with respect to enzyme concentration and incubation time. The reactions were stopped by the addition of 0.05 ml. of 30 per cent perchloric acid followed by dilution with 2 ml. of acetone. After centrifugation, the amount of red reduced TPTA in the supernatant solutions was measured at 540 rnp.

$ The activity on DMP was set arbitrarily at 160 in both assays.

TABLE IV E$ect of DMP Concentration on Glycine Reduction to Acetate

-SH molarity Acetate formed

0 0.6033 0.033 0.066 0.099

- wmdes Qn 0.5 ml.

0.12 0.30 1.61 1.56 1.32

Each sample contained orthophosphate 56 pmoles, ADP 2 rmoles, enzyme (Frac- tion B) 3.6 mg., and dimercaptan as indicated above. Other reactants are listed in Table I. Incubation time, 120 minutes.

968

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T. C. STADTMAN, P. ELLIOTT, AND L. TIEMANN 969

2, Table V, data are presented which show that both enzyme fractions were also needed for the arsenolysis reaction. Thus, one protein fraction was not merely supplying the enzymes necessary for the final transfer of phosphate to ADY. In contrast to results of previous experiments (Table II), the reconstructed system used for Experiment 1 (Table V) formed much more acid-labile phosphate than acetate; similar results were ob- tained with many other recombined Fractions E and F. Moreover, it was also observed that even higher ratios of phosphate esterified to acetate formed could be obtained with a given enzyme combination by increasing

TABLE V

Separation of Glycine Reduetaase System into Two Protein Components

Experiment X0. Protein fraction

E F E+F E F E+F

Phosphorolysis reaction

PIOllliIl. formed* “ste:

Jm7lcs pmmoles

0 0.05 0.19 0.11 1.35 0.76 0.24 0.03 0.42 0.42 1.80 1.61

-

Arsenolysis reaction

Acetate formed

pmolcr

Not measured “ ‘I “ “

0.39 0.47 1.76

In Experiment 1,2.1 mg. of Fraction E and 2.34 mg. of Fraction F were employed. Other components were those described in Table I together with KsHPO, 3 rmoles, AMP 6 pmoles, and ADP 0.1 @mole; final volume, 0.6 ml. Experiment 2 was similar to Experiment 1 except that different enzyme preparations were used. Fraction E was employed at a level of 1 mg. and Fraction F at 4.04 mg. In the phosphate-con- taining system there were K~HPOI 5 amoles, AMP 5 pmoles, and ADP 2 rmoles. In the arsenate system these reactants were replaced with NagHAsO, 10 rmoles.

* See Table II.

the concentration of the amino acid substrate (Table VI). Thus, at a concentration of 3.17 X lop2 M glycine, 4 times as much phosphate is es- terified as there is acetate formed. Although glycine disappearance could not be estimated at this high substrate concentration, it seemed likely that another product must have accumulated, the formation of which was coupled with the esterificat,ion of phosphate. Examination of reaction mixtures which contained glycine-Cl4 revealed that a labeled product, volatile from alkaline solution (pH about lo), was present.a Aliquots of the distillate, containing neither labeled acetate (counts stable to drying

* Samples, diluted to about 20 ml., were distilled in an all-glass microstill with a short reflux column; two-thirds of the total volume was collected in an ice-chilled receiver.

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970 ENZYMIC REDUCTION OF AMINO ACIDS. III

at alkaline pH) nor labeled glycine (counts stable to drying at acid or alkaline pH), when oxidized with acid chromate or acid ceric sulfate yielded Cl402 as the only detectable labeled product.4 There was approximately the same yield of labeled COz upon oxidation of distillates prepared from parallel reaction mixtures containing glycine-l-C14 or glycine-2-P. Thus both carbons of glycine appear in the volatile fraction and, since they are found in about equal amounts, it seems likely that they are in a single compound.

In a somewhat larger scale experiment, 4 pmoles of the volatile product (assuming 1 labeled carbon atom per mole) accumulated when 10 ccmoles of glycine-2-Cl4 and 17 pmoles of DMP were incubated for 90 minutes in a tot.al volume of 1.26 ml. with 2.6 mg. of Fract,ion E, 6.36 mg. of Fraction

TABLE VI Effect of Glycine Concentration on ATP: Acetate

Glycine concentration PM min. formed* Acetate formed

x pmles f.mwle

0 0.24 4.75 x 10-a 0.71 0.53 7.95 x 10-a 0.93 0.49 1.59 x 10-z 1.00 0.48 3.17 x lo-9 1.70 0.41

The enzyme was 1.31 mg. of Fraction E plus 3.18 mg. of Fraction F. Samples (0.63 ml.) were incubated anaerobically, 90 minutes.

* See Table II.

F, and doubled amounts of the other necessary components (Table I). In addition, 0.8 pmole of acetate accumulated, 2.7 pmoles of orthophos- phate disappeared, and 2.7 pmoles of acid-labile phosphate were formed. In view of the amount of phosphate esterification observed, it is tempting to suggest that the volatile compound may actually be a condensation

4 The labeled volatile product was oxidized with 0.9 N K&r*04 in 3 N H&SO4 (final concentrations) overnight at room temperature or for 30 minutes at 70”. The mix- ture was then steam-distilled slowly and the distillate collected in alkali. No la- beled volatile acid was found; only Cr40Z was collected. Control experiments with C”-labeled acetate showed only 30 per cent conversion to CrQ2 under these condi- tions; the remainder was recovered as unchanged acetate. Oxidation of the unknown with milder chromate (1 M CrOa in 0.7 N H&O,), under conditions whereby acetate was not attacked, also gave no labeled volatile acid and much lower yields of CWS (40 per cent or less). Ceric sulfate (0.66 saturated) in 0.66 N HaSO4 oxidized the unknown to CY402 as efficiently as did the stronger acid chromate when similar condi- tions were employed. In all cases the conversion to Cl402 occurred during the slow steam distillation and not during the preceding step.

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T. C. STADTMAN, P. ELLIO!l’“l’, AND L. TIEMANN 971

product of 2 glycine residues, the formation of which could have required 1 equivalent of esterified phosphate. Thus the total amount of phosphate esterified actually would have been 4.7 pmoles (2.7 pmoles net plus 2.0 pmoles consumed), an amount equivalent to the total glycine products formed (4.8 pmoles). Thus far, attempts to identify the volatile product have been unsuccessful. It has not yielded a dinitrophenylhydrazone de- rivative, nor is it retained on Dowex 50-H+. Also, as judged by the con- ditions of oxidation to COZ, it is probably not ethanol, acetaldehyde, di- acetyl, or acetyl methyl carbinol.

Reversibility of Reaction-Combined Fractions E and F as well as Frac- tion B were studied with respect to their ability to catalyze the reversal of Reaction 2. When high concentrations of reactants5 and prolonged incubation times were employed, only a slight conversion of acetate-C’* to a non-volatile compound was observed.

Inhibitors-Substances that have been found to inhibit the reductive deamination of glycine by enzyme preparations of C. sticklandii are NHzOH (100 per cent by 1O-2 M), sodium fluoride (50 per cent by 1.67 X 10” M),~

metals such as iron, manganese, and cobalt, when added in trace amounts, and atmospheric oxygen (50 to 75 per cent inhibition when air, 2.5 ml., instead of helium is the gas phase in 10 X 75 mm. stoppered tubes). The reaction is not sensitive to dinitrophenol (5 X 10m5 M) ; however, the &a- bility of dinitrophenol in these preparations has not been determined.

DISCUSSION

One of the most interesting facets of the glycine reductase system of C. sticklandii is the mechanism of ATP synthesis. In view of the fact that arsenate can replace, completely, orthophosphate and the adenylate nucleotide in this reaction, it is likely that a phosphorylated intermediate is formed normally and that this subsequently transfers its phosphate moiety to ADP to form ATP. The corresponding arsenate derivative presumably decomposes spontaneously.

Among the known high energy phosphate compounds that might be considered as possible intermediates are acetyl phosphate and phosphor- amidate. The former already is somewhat unlikely when one considers

6 The following concent.rations of reactants were used: potassium acetate-W, 100 pmoles (pH 5.5); (NH4)2SOa, 50 rmoles; ATP, 10 pmoles; oxidized lipoic acid, 2.5 pmoles (or 1,3-dithiopropanol, 2.5 pmoles); and MgC12, 10 pmoles in a total vol- ume of 1.0 ml. In other experiments phosphoenolpyruvate, pyruvate kinase, and ADP, as well as acetyl phosphate and ADP (acetyl kinase supplied in Fraction B), were used to generate ATP. No reaction was detected at pH 8.7.

E The same extent of inhibition was observed for the phosphorolyais reaction (1.67 X 10m2 M Pod’) and the arsenolysis reaction (1.67 X lO+ M AsO,‘). Mg++ (5 X 10ea M) was present in each instance.

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972 ENZYMIC REDUCTION OF AMINO ACIDS. III

that it is the a-carbon atom of glycine that undergoes reduction in its conversion to acetate. Moreover, in trapping experiments in which pu- rified transacetylase and substrate levels of CoA replaced AMP and ADP, there was no accumulation of acetyl CoA. No material that reacts with hydroxylamine to form a hydroxamate has been found to accumulate in complete reaction mixtures or those from which either one of the enzyme fractions or the nucleotide acceptor has been omitted. Also, removal of t,he active acetyl kinase from the extracts, by preparation of Fractions E and F, resulted in no loss of ability of the glycine reductase system to form aeetate and ATP. In preparations which contain a very active acetyl kinase, synthesis of ATP from acetyl phosphate and from the reductive deamination of glycine was additive.

Phosphoramidate can be excluded as the phosphorylated intermediate since this compound did not give rise to any ATP formation when incu- bated with the enzyme system. The only activity detected wm a slow hydrolysis to orthophosphate and ammonia by a phosphatase present in Fraction E and in the crude preparations. Likewise, no ATP formation was observed with adenosine 5’-phosphoramidate (21) although the cor- responding derivative of ADP may conceivably be involved; however, the objection to all of these particular N-P compounds is that the type of group transfer reaction which would be expected to form them should place an oxygen atom on the amino donor compound. In the case of glycine the product should be glycolic acid but this, at least as the free compound, has been found to be completely inert in the system.

The remaining obvious possibility is the mercaptan; i.e., the phosphoryl- ated intermediate may be S-phosphoryl dimercaptopropanol. Some prec- edents for such a suggestion are recent reports (22, 23) that S-phosphoryl CoA occurs as a high energy phosphorylated intermediate in other bio- logical systems. The synthesis of ATP by the glycine reductase system is apparently not due to SH- oxidation alone since enzyme preparations containing bot,h the glycine reductase and n-proline reductase activities form ATP only when glycine acts as electron acceptor for DMP oxidation. Although n-proline is actively reduced to b-aminovalerate, there is no associated phosphorylation observed with this substrate (14).

With some purified enzyme preparations a stimulation of glycine con- version to acetate by the addition of imidazole has been observed. This may perhaps be due to a slow catalysis by imidazole of the cleavage of an intermediate that normally participates in an enzyme-catalyzed reaction. If the enzyme necessary for this step has been made rate-limiting by the purification procedure, the non-enzymatic catalysis by imidazole might then be detectable.

Inhibition of glycine conversion t,o acetate by fluoride in the case of the

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T. C. STADTMAS, I’. ELLIOTT, AND L. TIEMANN 973

arsenolysis as well as the phosphorolysis reaction implicates a metal cat- alyst for botch processes.

The formation of acetate from glycine by 6. sticklandii results in no mixing of the carbon atoms; thus this process is completely distinct from that occurring in other glycine-fermenting organisms such as Diplococcus glycinophilus and Clostridium acidi-urici (24).

SUMMARY

Utilization of 1,3-dimercaptopropanol as electron donor has made pos- sible a more detailed study of the reductive deamination of glycine in a soluble system prepared from Clostridium sticklandii.

The partial purification and some of the properties of this glycine re- ductase system are described.

Balance experiments carried out with purified enzyme preparations show that, for each mole of glycine reduced to a mole each of acetate and am- monia, 2 equivalents of SH- are oxidized and 1 mole of orthophosphate is esterified.

BIBLIOGRAPHY

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10. Fiske, C. H., and Subbarow, Y., J. Biob. C&m., 66, 375 (1925). 11. Eggleston, L. V., and Hems, R., Biochem. J., 62, 156 (1962). 12. Peel, J. I,., and Loughman, B. C., Biochem. J., 66, 709 (1957). 13. Phares, E. F., Arch. Biochem. and Biopkys., 33, 173 (1951). 14. Stadtman, T. C., and Elliott, P., J. Biol. Chem., 996, 983 (1957). 15. Kunite, M., J. Gen. Physiol., 36, 423 (1952). 16. Stadtman, E. R., Novelli, G. D., and Lipmann, F., J. Biol. Chem., 191,365 (1951). 17. Kornberg, A., J. Biol. Chem., 163, 779 (1950). 18. Hager, L. P., and Gunsalus, I. C., J. Am. Chem. Sot., 76, 5767 (1953). 19. Cutolo, E., Arch. Biochem. and Biophys., 64, 242 (1956). 20. Stadtman, T. C., Biochem. J., 62, 614 (1956). 21. Chambers, R. W., Moffatt, J. G., and Khorana, H. G., J. Am. C&m. Sot., 79,

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TiemannThressa C. Stadtman, Patricia Elliott and Lydia

REDUCTIONCOUPLED WITH GLYCINE

PHOSPHATE ESTERIFICATIONREDUCTION OF AMINO ACIDS: III.

STUDIES ON THE ENZYMIC

1958, 231:961-973.J. Biol. Chem. 

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