Oxidative Inactivation of Carbamoyl Phosphate Synthetase (Ammonia)

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The two molecules of ATP used in the reaction have differ- ent roles and binding sites. ... We use here NaB3H4 to label carbonyl groups generated by the ...
Vol. 267, No. 7, Issue of March 5, pp. 4524-4532,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 hy The American Society for Biochemistry and Molecular Biology, Inc.

Oxidative Inactivationof Carbamoyl Phosphate Synthetase(Ammonia) MECHANISM AND SITES OF OXIDATION, DEGRADATION OF THE OXIDIZED ENZYME, AND INACTIVATION BY GLYCEROL, EDTA, AND THIOLPROTECTING AGENTS* (Received for publication, July 25, 1991)

Eulalia AlonsoS, Javier CerveraQll, AntonioGarcia-EspaiiaQ(1, Elena BendalaQ**,and Vicente Rubiojn From the flnstituto de Investigaciones Citoldgicas, F. I. B. (Centro Asociado del CSIC), Amudeo de Saboya, 4, 46010Valencia, Spain and the $Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universitat de Valencia, 46010 Valencia, Spain

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+,ascorbate, and 02,but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+,and catalasehave no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site forATPA(ATPAyields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOHterminal domains. The oxidative inactivation promoted by ATP is inhibited byMg2+,Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337.Groups in the 60-kDadomain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPBand support the binding of ATPB in the 60-kDadomain. We also demonstrate that mercaptoethanol, reducing impurities inglycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in theoxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.

Carbamoyl phosphate synthetase,the enzyme that controls

* This work was supported by Grants PB85-0198 and PB87-0189 from the Direccibn General de Investigacibn Cientifica y TBcnica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Members of the Instituto de Investigaciones Citolbgicas-Kansas University Medical Center International Molecular Cytology Program. 11 Fellow of the Institucio Valenciana d’Estudis i Investigacib. ** Severo Ochoa Fellowof the Ayuntamiento de Valencia.

the entryof ammonia into theurea cycle, catalyzes a complex reaction as follows. 2MgATP + HCOi

+ NH,

acetylglutamate, M e , K+

*

2MgADP + Pi + carbamoyl phosphate

(1) \-I

The two molecules of ATP used in the reaction have different roles and binding sites. ATPe’ provides the phosphoryl group of carbamoyl phosphate and binds to the enzyme with high affinity even in the absence of the allosteric activator, acetylglutamate (1-3). ATPA yields the Pi, binds to the enzyme with less affinity than ATPB, and, if acetylglutamate is not present, the affinity for ATPA decreases dramatically (13). Since the affinities for Mg2+ and K+ (the ionic activators of the enzyme) also decrease in the absence of acetylglutamate (3), it is likely that ATPA,M$+, and K’ bind in close association to thesame region of the enzyme (4). Our structural knowledge of carbamoyl phosphate synthetase is very limited, and, thus, the structural basis of these changes in affinity and, in general, of the changes associated with allosteric activation need to be characterized. Limited proteolysis studies have identified 4 compact domains in mammalian carbamoyl phosphate synthetase (a single polypeptide of 160 kDa) of approximately, counting from the amino terminus, 40, 40, 60, and 20 kDa (5-8). The aminoterminal domain of 40 kDa is homologous to thesmall subunit of the Escherichia coli enzyme (9). The remainder is homologous to thelarge subunit of the E. coli enzyme and is composed of two homologous halves joined in tandem (9). We showed that acetylglutamate binds to the COOH-terminal domain of 20 kDa (lo), and, that, in the E. coli enzyme, the allosteric inhibitor UMP binds to an equivalent COOH-terminal domain (11).We have concluded that this is the regulatory domain of carbamoyl phosphate synthetases (11).Post et al. (12) have identified residues, using directed mutagenesis, that are essential for the binding of the molecules of ATPA and ATPB, in the second domain of 40 kDa and in the 60-kDa domain, respectively. We proposed, to account for the effects of acetylglutamate on thebinding of ATPA and account to for The abbreviations used are: ATPB, the ATP molecule that provides the phosphoryl group of carbamoyl phosphate; ATPA,the ATP molecule that gives Pi in the reaction; AG and acetylglutamate, Nacetyl-L-glutamate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTNB, 5, 5’-dithiobis-(2-nitrobenzoic acid); 4-PyS2, 4,4’-dithiodipyridine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; TPCK, tosylphenylalanyl chloromethyl MOPS, 3 4 N ketone; FSBA, 5’-p-fluorosulfonylbenzoyladenosine; morpho1ino)propanesulfonicacid.

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Carbamoyl Phosphate Synthetase the regions of interaction between the small and the large subunit reported by Guillou et al. (13) in the E. coli enzyme, that the two regions of internal homology are associated as a pseudohomodimer in complementary isologous association (11).This folding allows interaction of the COOH-terminal domain with the adjacent domain of 60 kDa and with the second domain of 40 kDa. We proposed also that the regulatory domain is involved in facilitating the binding of ATPA and in closure of the binding site to allow formation of carboxyphosphate in the absence of water (10). We reportedrecently that carbamoyl phosphate synthetase is inactivated by a chemical model system that mimics mixedfunction oxidation systems and which consists of Fe3+,ascorbate, and oxygen (4). Enzymes found to be inactivated by mixed-function oxidation bind a divalent metaland generally require a nucleotide (14) (two conditions fulfilled by carbamoyl phosphate synthetase), suggesting that the oxidation of metal and/or nucleotide sites is important in the inactivation. We found that acetylglutamate greatly accelerates the rateof oxidative inactivation of carbamoyl phosphate synthetaseand that theaddition of MgATP prevents the inactivation in the presence of acetylglutamate (4). We therefore concluded that the site responsible for oxidative inactivation is infrequently exposed in the free enzyme, and that it is exposed more frequently upon binding of acetylglutamate (4), as expected for the ATPA and metal sites (3). We explained the protective effect of ATP by postulating that groups at or in the proximity of the site for ATPA are oxidized when acetylglutamate is present and ATP is absent, and that the binding ofATPA prevents their oxidation (4). Since a metal binding site is believed to play a key role in the inactivation by binding Fez+, we decided to examine the involvement of the metal site and other aspects of the mechanism of oxidative inactivation, including the location of the groups that experience oxidative modification. We use here NaB3H4 to label carbonyl groups generated by the oxidative treatment (15) and thiol reagents to study the reactivity of cysteines in theenzyme. Unexpectedly, in thecourse of these studies, we found that, in the absence of M e , ATP promotes the oxidative inactivation of the enzyme, and thatacetylglutamate does not need to be present.We conclude that theinactivation promoted by ATP is due to the oxidative modification of the ATPB site. Thus, this sitemay also be marked oxidatively. Although residues in the two ATP sites have been identified by directed mutagenesis (12), confirmation of the location of the ATP sites by a different technique is important. If, as proposed in our model for the folding of the enzyme (11),the two homologous regions of the enzyme are closely associated and the reaction takes place at the interface between these regions, it is conceivable that amino acid replacements in onehalf may influence functions in theother. Thus, theoxidative changes reported here provide an independent confirmation of the conclusions obtained from directed mutagenesis studies and may guide further mutagenesis work. Since oxidation may tag enzymes for degradation (16), we have also compared the susceptibility of the native and oxidatively inactivated forms of the enzyme to degradation by lysosomal or mitochondrial liver fractions. We also report that contaminants in glycerol and thatthiol protecting agents and EDTA, which are used frequently to protect enzymes (17), may promote the oxidative inactivation of carbamoyl phosphate synthetase. MATERIALS ANDMETHODS

Reagents-Solutions of 10 mMFeC13 (Fisher Scientific) in 1 mM HCl, of neutralized 1.25 M L-ascorbic acid (Sigma) in 50 mM Hepes, pH 7.2 (4), of 2 mM DTNB (5,5'-dithiobis(2-nitrobenzoic acid),

I Oxidation

4525

Sigma) in 0.1 M potassium phosphate buffer, pH 6.8, and of 6 mM 4PySz (4,4'-dithiodipyridine, Sigma) in dimethylformamide were prepared daily. Monobromobimane was obtained from Calbiochem and was dissolved at a concentration of 8 mM in acetonitrile. Glycerol (87% solution, analytical grade) was from Merck. Reducing impurities in the poly01were demonstrated by boiling for 5 min 20% (v/v) glycerol with 5 volumes of Benedict's reagent (18).N-(Chloroacety1)(285 Ci/mol) and N-acetyl-~-["C]glutamate (285 ~-['~C]glutamate Ci/mol) were synthesized as described previously (10, 19). Sodium [3H]borohydride(12.3 Ci/mmol) was provided by Du Pont-New England Nuclear and was dissolvedin 0.1 N NaOH. Other chemicals were of the highest purity available from Sigma, Merck, or Boehringer. Enzymes and Enzyme Assays-Rat liver carbamoyl phosphate synthetase (specific activity 29-32 pmol of carbamoyl phosphate/l5 min/mg) prepared essentially as described by Guthohrlein and Knappe (20) was stored a t -20 "C as an ammonium sulfate precipitate. Before use, it was dissolvedin the appropriate buffer, and itwas freed from ammonium sulfate by centrifugal gel filtration (21). Trypsin (bovine pancreas), catalase (bovine liver), and hexokinase and glucose-6-phosphate dehydrogenase (both from yeast) were from Boehringer Mannheim. TPCK-treated trypsin (from bovine pancreas), elastase (type IV, porcine pancreas), superoxide dismutase (from bovine blood), pyruvate kinase and lactate dehydrogenase (both from rabbit muscle), and protein standards for electrophoresis (High Molecular Weight Standard Mixture and Dalton Mark VII-L Mixture) were from Sigma. Carbamoyl phosphate synthetase activity was assayed spectrophotometrically a t 37 "C by coupling ADP production to NADH oxidation using pyruvate kinase and lactate dehydrogenase as described in Ref. 20, except for the omission of mercaptoethanol from the assay mixture. The HC03--dependent ATPase partial reaction was assayed in the same wayby replacing (NH4)zS04by&SO4 in the cuvette as described earlier (22). The partial reaction of ATP synthesis was assayed a t 37 'C by coupling the production of ATP to thereduction of NADP. The mixture (1ml) contained 50 mM glycylglycine,pH 7.4, 15 mM MgSOr, 5 mM ADP, 5 mM carbamoyl phosphate, 100 mM KCl, 10 mM acetylglutamate, 15 mM glucose, 0.75 mM NADP, 100 pg/ml hexokinase, and 2.5 pg/ml glucose-6-phosphate dehydrogenase. Unless indicated, protein was determined by the method of Bradford (23), using bovine serum albumin as standard. Oxidative Inactivation of Carbamyl Phosphate Synthetase-The inactivation was carried out at 37 'C essentially as described (4) in open tubes (12-mm diameter) containing the enzyme (2-10 mg/ml) in 0.05-0.2 ml of a solution containing 50 mM Hepes, pH 7.2, 0.1 M KCl, 0.25 mM FeC13, 31 mM ascorbate, and theadditions indicated in the tables and figures. The reaction was terminated by dilution (20200-fold) in the enzyme activity assay or by removal of the oxidative system by conventional or centrifugal gel filtration through Sephadex (G-25 or G-50) equilibrated with the appropriate buffer. Reaction with DTNB or with 4-PySz-The reaction with DTNB ) carried out in 25 mM Tris-HC1, pH 7.7,4 mM EDTA, 10 (20 p ~was mM KCl, and 10 mM acetylglutamate (24). The reaction with 4-PySz (5 pM) was carried out in 220 mM K-MOPS, pH 7.0,O.l mM EDTA, and 10 mM acetylglutamate (25). The enzyme, placed in these solutions by centrifugal gel filtration, was used a t a concentration of 0.5 mg/ml, and the temperature was30 'C in the two reactions. The change with time of the optical absorption at 412 (DTNB) or 323 nm (4-PySz) was monitored after the addition of the appropriate SHreagent. Controls without enzyme run in parallel were subtracted. Millimolar extinction coefficients for the reduced forms of DTNB and 4-PyS2 of 14.7 at 412 nm and 19.2 at 323 nm, respectively (determined with cysteine under identical experimental conditions), were used. The total number of SH groups per enzyme monomer was determined with 4-PySz in the presence of guanidinium chloride at pH 6.5 as reported (25). The concentration of the enzymewas determined by the biuret method (26) using bovine serum albumin as standard. Labeling with Monobromobimnne and Limited Proteolysis of the Labeled Enzyme-The procedure used was essentially that described in Ref. 27. The enzyme (1.3 mg/ml; intact or oxidatively inactivated) was placed in 50 mM Hepes, pH 7.6, 0.1 mM EDTA, by centrifugal gel filtration and was incubated for 15 min at 37 "C with 10 mM acetylglutamate. Then, 87 p M monobromobimane was added and the incubation was continued for 5 min. Unbound monobromobimane was removed bygel filtration through a Sephadex G-25 column (PD10 column, from Pharmacia LKB Biotechnology Inc.) equilibrated with 75 mM Hepes, pH 7.6, containing 1 mM acetylglutamate. The labeled enzyme (0.5-0.9 mg/ml) was incubated in the dark a t 37 "C

Carbamoyl Synthetase Phosphate

4526

with 2 pg/ml trypsin (TPCK-treated). After 60 min, 1 mM PMSF was added, the mixture was immediately frozen and lyophilized, and the residue, dissolved in 2 ml of 70% (v/v) formic acid, was applied to a column of Sephadex G-75 (90 X 1.5 cm) equilibrated and eluted with 10% (v/v) acetic acid. Fractions (6 ml) were collected, and the absorbance at 280 nm and the fluorescence (excitation wavelength, 395 nm; emission wavelength, 475 nm) were monitored. Samples from the UV-absorbing peaks were subjected to SDS-PAGE (28) in 7.5% polyacrylamide gels. Labeling of Carbamoyl Phosphate Synthetase with NaB3H4 and Limited Proteolysis of the Labeled Enzyme-The enzyme (2-3 mg/ml, intact or oxidatively inactivated) was placed in 0.1 M Tris-HC1, pH 8.5, 0.1 M KCl, using centrifugal gel filtration. 0.04-20 mM NaB3H4 (12.3-0.0246 Ci/mmol) and 2 mM EDTA were added, and themixture was incubated at 37 "C.After 30 min, the NaB3H4was removed, and the enzyme was placed in a solution of 35 mM Tris-HC1, pH 7.4, 8% glycerol, 1 mM dithioerythritol, 70 mMKC1, 25 mM NaCl, using centrifugal gel filtration (two times in sequence). Samples from the effluent were either precipitated with 10% trichloroacetic acid to determine the radioactivity incorporated in the protein or were subjected to limited proteolysis by incubation at 37 'C with 10 mM acetylglutamate and 10 pg/ml elastase or 4 pg/ml trypsin. After 20 min, the digestion was stopped by rapid mixing of a volume of the solution with 2 volumes of a solution a t 100 "C of 0.2 M Tris-HC1, pH 6.8,3.2% SDS, 16% mercaptoethanol, 17% glycerol, and 0.0015% bromphenol blue. Boiling was continued for 5 min. The samples were subjected to SDS-PAGE (28) in 7.5% polyacrylamide gels. Duplicate gels were used for fluorography (29) and for protein staining with Coomassie Blue. The results were analyzed by densitometry using an Ultroscan laser densitometer (from LKB). Degradation of Carbamoyl Phosphate Synthetase by Subcellular Components--Rat liver mitochondria treated with digitonin to minimize lysosomal contamination (30) were freeze-thawed 9 times before use to prevent problems of latency. Cytosol was obtained by high speed centrifugation (1 h,100,000 X g) of the postmitochondrial supernatant (30). The cytosolic proteins were placed in 20 mM Hepes, pH 7.25, 50 mM NaCl by gel filtration through Sephadex G-25 (PD10 column). Lysosomes prepared from rat liver (31) were freezethawed three times before use. Protein in the various fractions was determined by the biuret method in the presence of sodium deoxycholate (32). Intact or oxidatively inactivated'(residua1 activity, 4 2 % ) carbamoyl phosphate synthetase, placed in the appropriate buffer by centrifugal gel filtration, was incubated with the different fractions at 37 "C in closed 1-ml syringes without an air chamber. The incubation of the enzyme (1 mg/ml) with the cytosol (2.5 mg/ml) was carried out in 20 mM Hepes, pH 7.25, 50 mM NaCl. The incubation of the enzyme (2.5 mg/ml) with mitochondria (13 mgof protein/ml) was carried out in the same medium. Incubations of the enzyme (0.5 mg/ ml) with lysosomes (0.45 mgof protein/ml) were carried out in 50 mM NaC1, 10 mM MES buffer adjusted to pH 5.5 or pH 7. At the indicated periods of incubation, the mixtures were analyzed by SDSPAGE using 6% polyacrylamide gels. After Coomassie staining, the amount of protein in the band of 160 kDa (corresponding to the noncleaved enzyme) was determined by densitometry.

I Oxidation TABLE I Influence of M P ,acetylglutamate, and ATP on the oxidative inactivation of carbamoyl phosphate synthetase When used, M%+ was added (as the chloride) at a concentration of 10 mM in excess of that of the nucleotide. k is the first order rate constant for inactivation. k Additions

NoMg2+ h-'

None 0.30 2.52 3.30 Acetylglutamate (10 mM) 10.40 ATP (1mM) ATP (5 mM) 0.24 UTP (5 mM) 13.90 ATP (1mM), acetylglutamate (10 mM) ATP (5 mM), acetylglutamate (10 mM) UTP (5 mM), acetylglutamate (10 mM)

Mg2+

0.06

0.84 0.06 2.52

ATP (mM)

FIG. 1. Influence of ATP on the oxidative inactivation of carbamoyl phosphate synthetase in the absence of M e . k is the first order rate constant for inactivation. I

I

I

I

I

I

RESULTS

Influence of Mg2+and ATP on theOxidative Inactivation of Carbamoyl Phosphate Synthetase-The enzyme is oxidatively

inactivated with pseudo-first order kinetics (4),and the inactivation is accelerated by acetylglutamate and is prevented by mixtures of ATP, Mg+, and acetylglutamate ((4)and Table I). ATP promotes the inactivation in theabsence of acetylglutamate, particularly if Mg2+ is absent (TableI). In theabsence of M$+, mixtures of ATP and acetylglutamate additively promote the inactivation. The actions of ATP appear to be specific, for UTP has no effect. The plot of the rate constant( k ) for the inactivation of the enzyme in the absence of Mg2+ and acetylglutamate versus the concentration of ATP is hyperbolic, giving an apparent KO for ATP of approximately 30 PM (Fig. 1).This value is similar to the KD (20-30 PM) for ATPB in the absence of acetylglutamate (3). The rateof inactivation in the presence of acetylglutamate

FIG. 2. Influence of the concentration of Mg2+on the oxidative inactivation of carbamoyl phosphate synthetase in the presence of 1 mM ATP and 10 mM acetylglutamate.

and ATP decreases rapidly with increasing concentrations of Mg", until the concentration of this metal equals that of ATP (Fig. 2), suggesting that binding of MgATP is responsible for the major part of the protection. A further decrease in the ratewhen the concentration of Mg2+ is increased suggests that the binding of Mg2+ to divalent metal site(s) in the enzyme may also provide some additional protection. Differences between the Oxidative Inactivation of the Enzyme in the Presence of Acetylglutamate or i n the Presence of A T P (Table IZ)-The inactivation in both cases requires oxygen (traces of oxygen account for the residual inactivation under aninert atmosphere),and it is unaffected by the addition of superoxide dismutase or the radical scavenger,

Carbamoyl Phosphate Synthetase

I Oxidation

4527

TABLEI1 enzyme remains uncleaved when the enzyme was virtually Influence of additions on the oxidative inactivation of carbamoyl inactivated completely. phosphate synthetase Oxidative Inactivation of the PartialReactions-The ability Acetylglutamate (12.5 mM)or ATP (5 mM)was present in the of the enzyme to catalyze the complete and two partial reacincubation with the oxidative system. Carbamoyl phosphate synthe- tions that reflect separate phosphorylation steps (33, 34) is tase activity was determined at the beginning and after 15 min of incubation. Results are expressedas a percentage of the inactivation lost simultaneously(Fig. 4, lowerpanel) uponoxidation in the without additions (46% in the presence of acetylglutamate; 85% in presence of acetylglutamate. In contrast, with oxidative inpresence of ATP, the ATPase partial activity the presence of ATP). None of the additions had any substantial activation in the effect onthe enzyme activity when ascorbate andFeCL wereomitted. (which reflectsthe activationof HC03- by ATPA) islost much less rapidly than the complete reaction and the partialreacInactivation Additions or omissions tion of ATP synthesis(which reflects the phosphorylation of Acetylglutamate ATP carbamate by ATPB) (Fig. 4, upper panel).Theseresults % of oxidation differdepending on indicatethatthesites None 100 100 whether acetylglutamate or ATP are present and suggest that, Helium atmosphere 8 5 when ATP is present, the ATP site involved in the phosMannitol (0.1 M ) 112 102 phorylation of carbamate is oxidized preferentially. Superoxide dismutase (200units/ml) 101 105 Reactivation of Oxidized Carbamoyl Phosphate SynthetaseMnC12 (5 mM) 111 17 EDTA (1mM) 23 209 The enzyme inactivated in the presence of acetylglutamate is Catalase (30,000 units/ml) 112 42 reactivated partly by incubation, after removal of the oxidaH202(10mM; no Fe or ascorbate) 0 0 20 mM dithioerythritol (Fig. 5) or with tivesystem,with Ascorbate oxidase (no Fe; 2.3 m M as14 262 mercaptoethanol (data not shown), suggesting the oxidation corbate)" of enzyme thiols to disulfides. The concentration of dithioThe ascorbate solution was stirred for 30 min at 23 "C with 17 erythritol affects the rate but not the final amount of reactiunits/ml ascorbate oxidase(immobilizedin a spatula; from Boehringer) priorto mixing with carbamoylphosphate synthetase. Results vation. Irrespective of the extent of inactivation (see results are expressed as a percentage of the inactivation in the absence of for 30 and 60 min, Fig. 5), only about one-half of the activity ascorbate oxidase and in the presence of 2.3 mM ascorbate and 0.25 lost is regained by treatment with dithioerythritol. Thus, a mM FeC13(26% inactivation inthe presence of acetylglutamate;59% mechanism of oxidation E + A B, in which E is theactive inactivation inthe presence of ATP). form of the enzyme, A is a reactivable form, and B is an irreversibly inactivated form is therefore excluded. All of the 1 2 3 4 increase in the inactivation effected byEDTA in the presence of acetylglutamate is reversible (data not shown). b 20sp Inactivationintheabsence of acetylglutamate, with or " V 118without ATP, cannot be reversed by thiols, whether M e is 97.4present (Fig. 5) or absent (data notshown). 66QSL Oxidation of Cysteines 1327 and 1337 in the Presence of ." F -. Acetylglutamate-In the presence of acetylglutamate, two es4ssential thiol groups (24, 25), recently identified as cysteines 36 1, 1327 and 1337 (27), react rapidly with thiol reagents such as DTNB, with concomitant inactivation of the enzyme. Oxidative inactivation in the presence of acetylglutamate prior

+.

-*

aB

&

-

::z $

.-

1 .

FIG. 3. SDS-PAGE (8.8% polyacrylamide gel) of oxidatively inactivated carbamoyl phosphate synthetase. Track I, molecular weight standards (M, values are indicated at the margin). Track 2, intact carbamoyl phosphate synthetase. Tracks 3 and 4, carbamoyl phosphate synthetase oxidatively inactivated (1-h incubation) in the presence of 12.5 mM acetylglutamate or 50 p~ ATP, respectively. mannitol. Thus, the inactivation appears not to involve superoxide anions in the solution and may be due to free radicals generated at sites within theenzyme that are inaccessible to the scavenger. MnC12 reduces drasticallytheinactivation promoted by ATP but not that promoted by acetylglutamate, excluding in the lattercase the involvement of physiological Me2+sites. EDTA prevents the inactivation induced by ATP, but promotes further the inactivation induced by acetylglutamate. Catalase decreases the inactivation inducedby ATP, and, thus, HzO, is involved in thiscase. However, H202 itself is not responsible for this inactivation (shown by the addition of 10 mM H202,Table 11). Treatment with ascorbateoxidase 10 20 30 40 50 60 70 indicates that dehydroascorbate inactivatesthe enzyme in the TIME (MIN) presence of acetylglutamate but not in the presence of ATP. FIG. 4. Loss of carbamoyl phosphate synthetase activity. SDS-PAGE (Fig. 3) demonstrates no cross-linking or fragof carbamoyl phosphate synthetase activity (0)and of the partial mentation of the enzyme (Mr= 160,000) when the inactiva- Loss activities of HC03--dependent ATP hydrolysis (ATPase)(.) and of tion is promoted by acetylglutamate, whereas there is sub- ATP synthesis from carbamoyl phosphate and ADP (A) upon incustantial fragmentation when the inactivation is promoted by bation of the synthetase in the oxidative system in the presence of 5 ATP. Even in the latter case, however, a large fraction of the mM ATP (upperpanel) or 12.5 mM acetylglutamate (lowerpanel).

Carbamoyl Phosphate Synthetase I Oxidation

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-.- -.--.-

per enzyme monomer (titrated with 4-PyS2 in 6 M guanidinium chloride) decreased by 1.5 groups (from 18.5 to 17 groups per monomer) with oxidative inactivation in the presence of acetylglutamate. Treatment with dithioerythritol fully restored the SH group reactivity. Thus, only the two rapidreacting cysteines are oxidized with the inactivation in the presence of acetylglutamate. *"".&"-~- -~ Oxidative inactivation in the presence of ATP does not affect the rapid reaction, although there is some increase in the reactivity of the slow-reacting thiols (Fig. 6, lower panel). In the absence of acetylglutamate in the reaction medium, cysteines 1327 and 1337 of the intact enzyme become much less reactive (24, 25, 27). The same occurs when the enzyme 1 2 3 L 5 is inactivated oxidatively in the presence of ATP (Fig. 6, lower TIME (HOURS) panel). Thus, the enzyme, inactivated under theseconditions, FIG.5. Partialreactivation by dithioerythritol (Dl") of oxidatively inactivated carbamoylphosphate synthetase. The binds acetylglutamate and undergoes the same conformaenzyme was incubated with the oxidative system containing MgCll tional changes (as far as SH group reactivity is concerned) as (10 mM in excess of the nucleotide) a n d 0, no additions; 0, 10 mM the normal enzyme. ATP; A, 12.5 mM acetylglutamate; 5 mM ATP and 12.5 mM Although the rapidly reacting thiols are not oxidized when acetylglutamate. After the period of incubation a t 37 'C indicated by the enzyme is inactivated in the presence of ATP, titration the arrows, the enzyme was subjected to centrifugal gel filtration through Sephadex G-50equilibrated with 125 mM glycyl-glycine,pH with 4-PyS2 in 6 M guanidinium chloride demonstrates the 7.4, and 100 mMKC1. Then, 20 mM dithioerythritol was added, and loss of 1.4 SH groups per monomer. The location of these the incubation at 37 "C was continued. The results are corrected for groups and their role in the inactivation remain to be deterprotein losses during centrifugal gel filtration (