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Clostridial Glycine Reductase: Protein C, the Acetyl Group Acceptor

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Clostridial Glycine Reductase: Protein C, the Acetyl Group Acceptor Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7853-7856, October 1989 Biochemistry Clostridial glycine reductase: Protein C, the acetyl group acceptor, catalyzes the arsenate-dependent decomposition of acetyl phosphate (acetyl ester 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 acetate 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 thiol-reducing agent threitol, and phosphotransacetylase from Clostridium and Mg2+ are required for catalysis 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 product 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 enzyme 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 proteins 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 thiols 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.
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