Inactivation of the Amidotransferase Activity of Carbamoyl Phosphate Synthetase by the Antibiotic Acivicin*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 6, Issue of February 8, pp. 4368–4373, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Inactivation of the Amidotransferase Activity of Carbamoyl Phosphate Synthetase by the Antibiotic Acivicin* Received for publication, September 6, 2001, and in revised form, November 27, 2001 Published, JBC Papers in Press, November 29, 2001, DOI 10.1074/jbc.M108582200 Bryant W. Miles‡, James B. Thoden§, Hazel M. Holden§¶, and Frank M. Raushel‡ʈ From the ‡Department of Chemistry, Texas A & M University, College Station, Texas 77842-3012 and the §Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Carbamoyl phosphate synthetase (CPS) from Esche- such as hallucinations, ataxia, paranoia, amnesia, hostility, richia coli catalyzes the formation of carbamoyl phos- and transient unconsciousness (9, 10). These neurological side phate from 2 mol of ATP, bicarbonate, and glutamine. effects take 24–72 h to manifest themselves and do not corre- CPS was inactivated by the glutamine analog, acivicin. late with peak levels of acivicin, suggesting that the drug is In the presence of ATP and bicarbonate the second- metabolized into a neurotransmitter antagonist (4). order rate constant for the inactivation of the glu- ؊1 ؊1 4 tamine-dependent activities was 4.0 ؋ 10 M s .In the absence of ATP and bicarbonate the second-order rate constant for inactivation of CPS was reduced by a factor of 200. The enzyme was protected against inactivation by the inclusion of glutamine in the reaction mixture. The ammonia-dependent activities were unaffected by the incubation of CPS with acivicin. These results are consistent with the covalent labeling of the glutamine-binding site located within the small amido- STRUCTURE 1 transferase subunit. The binding of ATP and bicarbonate to the large subunit of CPS must also induce a Rapidly proliferating tumor cells have elevated activities of the conformational change within the amidotransferase glutamine amidotransferases involved in purine and pyrimi- domain of the small subunit that enhances the nucleo- dine biosynthesis. Consequently, these cells consume signifi- philic character of the thiol group required for gluta- cant amounts of glutamine and deplete this amino acid from mine hydrolysis. The acivicin-inhibited enzyme was the blood (4, 5, 11, 12). Acivicin is a glutamine antagonist that crystallized, and the three-dimensional structure was irreversibly inactivates many of the glutamine amidotrans- determined by x-ray diffraction techniques. The thiol ferases including formylglycinamidine ribonucleotide synthe- group of Cys-269 was covalently attached to the dihy- tase (13), 5-phosphoribosyl 1-pyrophosphate amidotransferase droisoxazole ring of acivicin with the displacement of (14), CTP synthetase (15), GMP synthetase (16), and CPS1 a chloride ion. (17–20). All of the glutamine amidotransferases possess an essential cysteine residue that initiates an attack on the amide carbonyl of glutamine to form a covalent ␥-glutamyl thioester Acivicin (I) is a heterocyclic analog of glutamine (II) with intermediate. Acivicin contains a 4,5-dihydroisoxazole ring anti-cancer and anti-tumor activity that was initially discov- substituted with a chloride that is readily displaced (21–23). ered by screening fermentation broths of Streptomyces sviceus The activated thiolate anion of the essential cysteine residue (1–3). Administration of acivicin stops tumor growth in leuke- within the amidotransferase family of enzymes has been shown mia, melanoma, and a variety of carcinomas (4, 5), and contin- with some of these enzymes to covalently react with the di- ued administration of acivicin reduces tumor size and restores hydroisoxazole ring of acivicin, resulting in irreversible muscle mass. Unfortunately, acivicin also causes many unde- inactivation. sirable gastrointestinal and neurological side effects. The neu- The inactivation by acivicin of IGP synthetase (24), GMP rotoxic effects at low concentrations of acivicin include leth- synthetase (25), CPS II (26), CPS III (27), anthranilate synthe- argy, depression, insomnia, headaches, confusion, mood tase, and glutamate synthase (28) has been described. Acivicin swings, vivid dreams, and “large thoughts” (6–8). Higher con- stoichiometrically inactivates the glutamine-dependent activi- centrations of acivicin produce more severe neurological effects ties of IGP synthetase, GMP synthetase, CPS III, and anthranilate synthetase by alkylation of the nucleophilic cysteine residue located within the active site of these enzymes (24, 25, * This work was supported in part by National Institutes of Health 27, 28). The ammonia-dependent activities are unaffected by Grants DK30343 (to F. M. R.) and GM55513 (to H. M. H.). The costs of publication of this article were defrayed in part by the payment of page the covalent modification with acivicin, and the rate of inacti- charges. This article must therefore be hereby marked “advertisement” vation is facilitated by the presence of co-substrates. For exam- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ple, the addition of ATP and xanthosine monophosphate The atomic coordinates and structure factors (code 1KEE) have been greatly accelerates the rate of inactivation of GMP synthetase deposited in the Protein Data Bank, Research Collaboratory for Struc- tural Bioinformatics, Rutgers University, New Brunswick, NJ by acivicin (25). (http://www.rcsb.org/). CPS of Escherichia coli is a heterodimeric enzyme that cat- ¶ To whom correspondence may be addressed. E-mail: hazel_holden@ alyzes the formation of carbamoyl phosphate from bicarbonate, biochem.wisc.edu. ʈ To whom correspondence may be addressed: Dept. of Chemistry, P.O. Box 30012, Texas A & M University, College Station, TX 77842- 1 The abbreviations used are: CPS, carbamoyl phosphate synthetase; 3012. E-mail: [email protected]. IGP, imidazole glycerol phosphate. 4368 This paper is available on line at http://www.jbc.org Inactivation of CPS by Acivicin 4369 mate concentration was calculated using an extinction coefficient for Ϫ the reduced form of 3-acetylpyrimidine adenine nucleoside of 8.3 mM 1 cmϪ1 at 363 nm. The rate of glutamate formation when ATP and bicarbonate were present was determined using the same assay solution but supplemented with the addition of 5.0 mM ATP, 10 mM MgCl2, 10 mM ornithine, and 40 mM bicarbonate. The rate of ADP formation was assayed using a pyruvate kinase/ lactate dehydrogenase coupled assay by following the loss of NADH spectrophotometrically at 340 nm (35). The assay solution consisted of 0.1 M Hepes (pH 7.6, 25 °C), 1.0 mM phosphoenol pyruvate, 0.3 mM NADH, 0.1 M KCl, 20 mM ornithine, 20 mM MgCl2,10mM ATP, and 33 ␮g of pyruvate kinase and lactate dehydrogenase. For the glutamine- dependent utilization of ATP, 10 mM glutamine was added to the assay solutions. Inactivation of CPS by Acivicin—The rate of inactivation of the glutaminase and glutamine-dependent ATPase activities of CPS by acivicin was determined by incubating the enzyme (20 ␮M)in0.1M Hepes buffer (pH 7.6, 25 °C) with varying amounts of the inhibitor. At various time intervals, the aliquots were removed and diluted at least 100-fold into the appropriate assay solution. The time course for the fraction of the initial catalytic activity remaining (f) was a pseudo-first- order decay, and the rate constant k was obtained by a fit of the data to Equation 1. The plots of the pseudo-first-order rate constants versus the initial acivicin concentration yielded second-order rate constants for the inactivation of CPS. f ϭ eϪkt (Eq. 1) Effect of ATP and Bicarbonate on the Rate of Acivicin Modification of FIG.1. Ribbon representation of an ␣,␤-heterodimer of CPS. CPS—The rate of inactivation of the glutamine-dependent ATPase The small subunit is displayed in magenta. The large subunit is color- activity of CPS by acivicin and the rate of activation of the bicarbonate- coded in green, yellow, blue, and red to indicate the carboxy phosphate, dependent ATPase activity of CPS in the presence of 10 mM ATP and 40 the oligomerization, the carbamoyl phosphate, and the allosteric sub- mM bicarbonate were investigated. For the inactivation of the glutamine- domains, respectively. The tunnel connecting the three active sites is dependent ATPase activity, the time courses for the fraction activity displayed in a wire mesh representation. remaining were pseudo-first-order and were analyzed as described pre- viously. The time course for acivicin activation of the bicarbonate-dependent ATPase activity was also pseudo-first-order, and the rate con- glutamine, and two molecules of ATP. The synthesis of carbam- stant k was obtained by a fit of the data to Equation 2. In this equation oyl phosphate is accomplished via four separate reactions oc- v is the observed velocity, A is the amplitude factor, k is the rate curring at three physically distinct active sites connected by a constant of activation, and vo is the velocity observed when no acivicin tunnel that is ϳ100 Å in length and through which intermedi- is added. A plot of the pseudo-first-order rate constants as a function of ates are translocated (29, 30). As indicated in Fig. 1, the tunnel the acivicin concentration yielded the second-order rate constant for begins at the glutamine-binding site within the small subunit activation. and runs through the interior of the large subunit where it ϭ ͑ Ϫ Ϫkt͒ ϩ connects with the two binding sites for ATP. Cys-269 of the v A 1 e vo (Eq. 2) small subunit has been shown to function as the nucleophile Partition Ratio for Acivicin Inactivation of CPS—The partition ratio that attacks the amide carbonyl of glutamine to form the ␥-glu- for the inactivation of CPS with acivicin was determined. In these tamyl thioester intermediate. The ammonia is subsequently experiments CPS (20 ␮M) was incubated at 25 °C with various concen- channeled to the large subunit (31–33).

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