Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7853-7856, October 1989 Biochemistry Clostridial reductase: C, the acetyl group acceptor, catalyzes the arsenate-dependent decomposition of acetyl phosphate (acetyl intermediate/arsenolysis/glycine reduction/Clostidum sticklandii) THRESSA C. STADTMAN Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 108, Bethesda, MD 20892 Contributed by Thressa C. Stadtman, July 31, 1989

ABSTRACT The highly purified protein C component of action chromatography, first on phenyl-Sepharose and then clostridial glycine reductase is required in addition to seleno- on octyl-Sepharose columns. Molecular sieve chromatogra- protein A and protein B for the conversion of glycine to phy on Sepharose CL-6B at low ionic strength in the presence and ammonia in the presence of arsenate. As shown by of phosphate and then at higher ionic strength in the absence Arkowitz and Abeles [Arkowitz, R. A. & Abeles, R. H. (1989) of phosphate served as the final steps for isolation of protein Biochemistry 28, 4639-4644], the products are ammonia and C. Details of this procedure are to be published elsewhere. acetyl phosphate in the presence of phosphate. The protein C Acyl carrier protein and crystalline acetate kinase from component alone catalyzes an arsenate-dependent decomposi- Escherichia coli and iodoacetic acid were purchased from tion of acetyl phosphate, showing that it serves as the acetyl Sigma. Acetylphosphate as lithium/potassium salt, dithio- group acceptor in the overall reaction. A -reducing agent threitol, and phosphotransacetylase from Clostridium and Mg2+ are required for of the arsenolysis reaction kluyveri were from Boehringer Mannheim. by protein C. Alkylation or heating at 60C completely abol- The extent of acetyl phosphate decomposition in reaction ishes the ability ofprotein C to catalyze the arsenolysis reaction mixtures (0.84 ml) was measured after conversion of the and to participate as an essential component in the overall residual acetyl phosphate to acetylhydroxamate by addition glycine reductase reaction. of 0.16 ml of 4.5 M NH2OH. After 2 min the samples were acidified with 1 ml of 10% trichloroacetic acid, 4 ml of FeCl3 reagent (1.66% FeCl3-6 H20 in 1 M HCl containing 4% Glycine is used as a terminal electron acceptor by certain trichloroacetic acid) was added, and the iron-hydroxamate amino acid-fermenting (1, 2) and purine-degrading (3) anaer- complex was measured at 540 nm. obic bacteria. The reductive deamination ofglycine to acetate Alkylated protein C was prepared by reaction of the and ammonia is an exergonic process that is coupled to the reduced protein with 20 mM potassium iodoacetate. Solid synthesis of ATP (2, 4). The direct of the reaction KBH4 was added to the protein in 0.1 M Tricine-KOH buffer was shown recently (5) to be acetyl phosphate which, in the (pH 8), and the solution was incubated under argon for 30-40 presence ofADP, is converted to acetate and ATP by acetate min. Iodoacetate was added, and after 15-20 min at room kinase. The highly active acetate kinase, even when present temperature under argon, the reaction was quenched by as a minor contaminant of fractions, allows the addition of 2-mercaptoethanol to 40 mM. The solution was glycine reductase reaction to proceed in the unfavorable concentrated on an Amicon Centricon-30 microfilter at 40C, direction of acetyl phosphate generation by continuously and the protein was repeatedly washed with 50 mM converting the product to acetate and ATP. Alternatively, Tricine-KOH buffer (pH 7.5) to remove all reagents. Reduc- under in vitro conditions, the unfavorable equilibrium of the tion ofprotein C with borohydride followed by concentration reaction leading to acetyl phosphate synthesis can be shifted on Centricon-30 filters repeatedly has been shown to cause by the substitution of arsenate for phosphate. Spontaneous no loss of protein C activity, provided excessive foaming hydrolysis of the unstable arsenate ester (6) thus allows during KBH4 treatment is avoided. continued turnover of the acetyl group acceptor. In the The Bio-Rad protein assay standardized with bovine serum present report, the protein C component of the glycine albumin was used to estimate protein in partially purified reductase complex is identified as the acetyl group acceptor. protein C preparations. The finding that highly purified protein C catalyzes the arsenate-dependent decomposition of acetyl phosphate shows that an acetyl enzyme intermediate indeed is gener- RESULTS AND DISCUSSION ated. Reaction of this acetyl enzyme with arsenate in a The activity ofa glycine reductase complex reconstituted from back-reaction forms acetyl arsenate, which reacts with water highly purified selenoprotein A and protein C preparations and decomposes spontaneously (6, 7). This process is anal- (added in excess) and partially purified protein B, the carbonyl ogous to the arsenolysis of acetyl phosphate catalyzed by group component (4), was stimulated 2-fold by the addition of phosphotransacetylase (7). ADP and 3-fold by the substitution of arsenate for phosphate plus AMP and ADP (Table 1). Although each of the three MATERIALS AND METHODS enzyme fractions singly exhibited barely detectable acetate kinase activity, the amount supplied was sufficient to account The protein C component of glycine reductase was purified for the observed stimulation of glycine reduction in the pres- from sonic extracts of Clostridium sticklandii (8, 9) by using ence of added ADP. Under the same conditions, there was no a series of chromatographic steps. Preliminary fractionation increase in the amount of acetate produced upon further of the crude extract by ion-exchange chromatography on supplementation with pure E. coli acetate kinase (data not DEAE-cellulose (8, 9) was followed by hydrophobic inter- shown). In the experiment of Table 1, the protein B prepara- tion was the source of a small amount of phosphate, thus The publication costs of this article were defrayed in part by page charge accounting for the formation of 0.67 ,umol of product in the payment. This article must therefore be hereby marked "advertisement" absence ofadded phosphate. The reduced product (1.93 ,mol) in accordance with 18 U.S.C. §1734 solely to indicate this fact. of glycine formed in the absence of added adenylates was not 7853 Downloaded by guest on September 27, 2021 7854 Biochemistry: Stadtman Proc. Natl. Acad. Sci. USA 86 (1989)

Table 1. Glycine reduction to acetate by glycine reductase 5 complex reconstituted from purified A, B, and C 1.4

Acetate produced, ~1.2- Supplement added ,Umol None* 0.67 cio Phosphate E 20 mM 1.93 + ADP (8 mM)/AMP (8 mM) 4.1 06- Arsenate c0) 0.8 5 mM 6.0 a0) 10 10 mM 5.87 .L 0.6 *Reaction mixtures (0.5 ml) contained additions indicated above and 60 mM Tricine KOH (pH 8), 40 mM glycine, 80 mM (NH4)2SO4, 10 0.0 mM MgCI2, 40 mM dithiothreitol, ca. 100 ig of protein A, partially purified protein B (174 pg of protein), and highly purified protein C 0.005 10 15 20 25 30 (28 .g of protein). After incubation under argon for 90 min at 34WC, Time, min the reaction was terminated by addition of0.16 ml of4.5 M NH2OH. Acetate was estimated as acetyl hydroxamate after reaction with FIG. 1. Time course of arsenate-dependent decomposition of ATP and acetate (10, 11). acetyl phosphate. Reaction mixtures (0.84 ml) containing 50 ,umol of Tricine-KOH (pH 7.5), 10 umol of potassium arsenate (pH 7.5), 3 identified as acetyl phosphate but rather was assayed as acetyl ,mol ofacetyl phosphate, 10 ,mol ofMgCl2, 5 ,umol ofdithiothreitol, hydroxamate after reaction with added acetate kinase, ATP, and protein C (1.4 ug estimated from absorbancy at 277 nm; 2.2 pg and hydroxylamine (10, 11). Since the enzyme preparations by Bio-Rad protein assay) were incubated at 34°C under argon for the were not treated to remove traces of nucleic acid and bound indicated times. o, Total acetyl phosphate decomposed; e, acetyl nucleotides, the amount ofacetyl phosphate accumulated may phosphate decomposed in the absence of enzyme; A, enzyme- have been considerably less than 1.93 pumol. In view of the catalyzed acetyl phosphate decomposition (corrected for spontane- marked stimulation ofthe reaction by arsenate, it appears that ous rate). turnover ofthe acetyl enzyme intermediate is the rate-limiting mined under standardized conditions, with the indicated step ofthe glycine reductase reaction. To determine which one amount of this enzyme preparation is shown in Fig. 1. In of the glycine reductase protein components serves as the addition to arsenate, Mg2+ and such as dithiothreitol are acetyl group acceptor, proteins A, B, and C were assayed in required for decomposition of acetyl phosphate by protein C combinations for the to the arsenate- various ability catalyze (Table 3). With a dithiol as reducing agent (Fig. 2), a final dependent decomposition of acetyl phosphate. In the prelim- inary experiment (experiment 1 of Table 2), all of the acetyl concentration of about 6 mM appears to be optimal. Mercap- phosphate was decomposed in samples that contained protein toethanol was less effective at comparable thiol concentra- C, whereas with protein B alone or protein B plus protein A, tions. Based on previous observations that alkylation of pro- the loss of acetyl phosphate was similar to the amount de- tein C destroys its activity as an essential component of the composed spontaneously. As shown in experiment 2 ofTable glycine reductase complex (unpublished data), the effect of 2, only protein C was required for acetyl phosphate decom- treatment with iodoacetate on the ability of protein C to position, and the reaction was complete in 35 min or less with catalyze the decomposition ofacetyl phosphate was tested. As either 27.7 ,ug or 55.4 ,Mg ofenzyme added. Later, it was found shown in Table 4, the alkylated protein C exhibited negligible that 2.8 Ag of this highly active protein C preparation was still activity alone but, surprisingly, was markedly stimulatory in excess ofthe amount needed to decompose 3 tkmol ofacetyl when added to a comparable amount ofnative protein C. Heat phosphate in 30 min. The time course of the reaction, deter- treatment of the alkylated protein sample abolished its stim- ulatory effect (Table 4, experiment 2). Native protein C, Table 2. Acetyl phosphate decomposition by glycine reductase heated at 62-64°C under the same conditions, was also inac- protein components in the presence of arsenate tivated. The same degree of heat sensitivity was observed Protein component added Acetyl phosphate earlier when protein C was assayed for activity as a component of the reductase The Protein A Protein B Protein C decomposed, glycine complex (unpublished data). indication from these results that protein C may consist oftwo (ca. SO ,ug) (58 ,ug) (55.4 ,ug) ,umol components is further suggested by the nonlinear enzyme Experiment 1 concentration curves of Fig. 2. + + + 2.99 + + - 0.92 Table 3. Omission experiment - + - 0.93 Acetyl phosphate - + + 2.93 Reaction mixture decomposed, - - - 0.76* component omitted ,umol Experiment 2 - + + 2.95 None* 2.74 _ + +t 2.95 Arsenate 0.33 - - + 2.95 Dithiothreitol 0.44 MgCl2 1.18 Samples were incubated at 34WC under argon for 120 min (exper- iment 1) and 35 min (experiment 2). Reaction mixtures (0.84 ml) Enzyme 0.33t contained 50 ,umol of Tricine'KOH (pH 7.5), 3 ,umol of acetyl *The complete reaction mixture (0.84 ml) contained 50 ,umol of phosphate, 10 ,umol of MgCl2, 5 ,umol of dithiothreitol, 10 mol of Tricine-KOH (pH 7.5), 3 ,umol of acetyl phosphate, 10 ,umol of KH2AsO4, and the indicated proteins. The final pH was 6.8. potassium arsenate (pH 7.5), 10 ,mol of MgCl2, 1 ,mol of dithio- *The amount of acetyl phosphate decomposed in enzyme samples threitol, and 19 pg of protein C preparation. Samples were incu- lacking arsenate was the same as the amount that decomposed bated 40 min at 34°C under argon. spontaneously in the absence of enzyme. tAmount of acetyl phosphate hydrolyzed spontaneously under the tEstimation of 27.7 Ag from absorbancy of protein C at 277 nm. experimental conditions used. Downloaded by guest on September 27, 2021 Biochemistry: Stadtman Proc. Natl. Acad. Sci. USA 86 (1989) 7855

2 n E

-91.5- Cn -C5 0 ou

0)50.

.0 20 40 60 10 20 30 40 50 Protein C added, ,ul Protein C added, ,ul FIG. 2. Effects of varying enzyme and dithiothreitol concentra- FIG. 3. Arsenate-dependent decomposition of acetyl phosphate tions on acetyl phosphate decomposition. Reaction mixtures con- as a function of enzyme composition. Samples contained the indi- taining the indicated amounts of a protein C preparation [3 Mug of cated aliquots ofa diluted protein C preparation [9.4 Mug of protein per protein per 10 ,p1 (absorbancy at 277 nm) or 6.5 pg per 10 Mul (Bio-Rad 100 Mul of 50 mM Tricine-KOH (pH 7.5)]. o, Native protein C; A, protein assay)] and 1.2mM (o), 2.4mM (A), or 6 mM(o) dithiothreitol native protein C plus 10 ,ul of alkylated protein C (2.8 Mg of protein) were incubated with other components given in the legend of Fig. 1 added to each sample. The alkylated protein C was prepared as for 30 mm at 34°C under argon. described except that the reduced protein was allowed to react with 20 mM potassium iodoacetate for 1 hr. Other reagents were as described in the legend to Fig. 1 except that each sample contained A similar nonlinear response to enzyme concentration 10 Mmol of dithiothreitol. Incubation was for 30 min at 34°C under using a highly purified protein C preparation is shown in Fig. argon. 3. When parallel reaction mixtures were supplemented with a sample of the same enzyme preparation that had been Enzyme X acetate + arsenate inactivated by treatment with iodoacetate, the amount of enzyme X + acetyl arsenate, [2] acetyl phosphate decomposed was then a linear function of the amount of native protein C added (Fig. 3). In view of the Acetyl arsenate + H20 -* acetate + arsenate, [3] reactions presumably involved in the arsenate-dependent decomposition of acetyl phosphate (reactions 1-3), only one enzyme should be required since reaction 3, the hydrolysis of acetyl arsenate, is known to be spontaneous. Enzyme X + acetyl phosphate - The requirement of an added thiol and the inhibition of the enzyme X acetate + phosphate, [1] catalytic activity by alkylation suggest that an acetyl is generated on the enzyme which in the back-reaction then reacts with arsenate. The role of an additional heat-labile Table 4. Inactivation of protein C by alkylation and by component, not affected by alkylation, is thus not obvious. heat treatment Neither coenzyme A nor acyl carrier protein appears to Protein C added function in the reaction as the postulated thiol component. Acetyl phosphate No coenzyme Heated Heated decomposed, A was detected in the protein C preparations Native native* Alkylated alkylated* MLmol by assay with phosphotransacetylase (7). Addition of re- duced coenzyme A or acyl carrier protein from E. coli failed Experiment 1 to restore activity to alkylated protein C or to stimulate the 60Mg 1.56 activity of various levels of native protein C. 120ug - 2.46 An alternative explanation of the anomalous behavior of 60ug 0.03 the protein C preparations may be provided by the observa- 120Mug - 0.47 tion that the highly purified enzyme behaves as an associat- 60ug 60/.g 2.42 ing-dissociating system when subjected to polyacrylamide Experiment 2 gel electrophoresis or molecular sieve chromatography. It 60Mg - - 1.38 was observed repeatedly that, in the presence of 50 mM 60Mug 0.26 potassium phosphate (pH 7.2) and 1 mM dithiothreitol, the 60MAg 0.18 elution position of the protein from Sepharose CL-6B col- 60Mug 60ug 2.22 umns corresponds to Mr 400,000, whereas in 200 mM 60Mg 60 ig 1.44 CHES-HCl buffer [pH 8.7; CHES = 2-(N-cyclohexylami- Values in experiment 1 (10 Amol of dithiothreitol added) are no)ethanesulfonic acid] or in 50 mM Tricine-KOH buffer (pH corrected for 0.4 umol of acetyl phosphate decomposed spontane- 8) containing 2 mM dithiothreitol and 1 M , the protein ously. Therefore, for values of 2.42 and 2.46, the was was eluted from the Sepharose CL-6B column just after completely exhausted. In experiment 2 (1 Mmol of dithiothreitol catalase (Mr 240,000), indicating a Mr 200,000. When added), the values are corrected for spontaneous loss of 0.3 Mumol of rechromatographed on the same Sepharose CL-6B column in acetyl phosphate. Protein C (60 Ag of protein added in 50 mM 50 mM potassium phosphate, pH mM Tricine-KOH at pH 7.5) was a partially purified preparation eluted 7.2/1 dithiothreitol, the from a Sepharose CL-6B matrix. Other reactants are given in the Mr 200,000 species again emerged as a high molecular weight legend to Table 3. Reaction mixtures were incubated 30 min at 34°C species with the same profile observed initially. On native under argon. The alkylated protein was prepared as described. polyacrylamide gels, the protein migrated as two dissimilar, *The protein samples were heated in the reaction tubes in an equal smearing protein bands that partially overlap. One was volume of water for 10 min at 62-64°C prior to the addition of estimated to be slightly larger than Mr 100,000, and the other, reaction mixture components. slightly smaller. The indication that protein C is composed of Downloaded by guest on September 27, 2021 7856 Biochemistry: Stadtman Proc. Natl. Acad. Sci. USA 86 (1989)

two dissimilar, easily dissociable subunits suggests two ob- 4. Tanaka, H. & Stadtman, T. C. (1979) J. Biol. Chem. 254, vious possibilities: (i) one of the subunits has a regulatory 447-452. function or (ii) the catalytic site is formed at the interface 5. Arkowitz, R. A. & Abeles, R. H. (1989) Biochemistry 28, 4639-4644. between the two dissimilar subunits in the heterodimer. Ifboth 6. Stadtman, E. R. & Barker, H. A. (1950) J. Biol. Chem. 184, polypeptides are indeed essential components of fully active 769-793. protein C, then dissociation at very low protein concentrations 7. Stadtman, E. R. (1952) J. Biol. Chem. 196, 527-534. could account for the nonlinearity observed in Fig. 3. 8. Turner, D. C. & Stadtman, T. C. (1973) Arch. Biochem. Bio- phys. 154, 366-381. 1. Stickland, L. H. (1934) Biochem. J. (London) 28, 1746-1759. 9. Stadtman, T. C. (1978) Methods Enzymol. 53, 373-382. 2. Stadtman, T. C., Elliott, P. & Tiemann, L. (1958) J. Biol. 10. Sliwkowski, M. X. & Stadtman, T. C. (1988) BioFactors 1, Chem. 231, 961-973. 293-296. 3. Durre, P. & Andreesen, J. R. (1983) J. Bacteriol. 154, 192-199. 11. Rose, I. A. (1955) Methods Enzymol. 1, 591-595. Downloaded by guest on September 27, 2021