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Carbamoyl Phosphate Synthetase: a Tunnel Runs Through It Hazel M Holden*, James B Thodent and Frank M Raushel

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Carbamoyl Phosphate Synthetase: a Tunnel Runs Through It Hazel M Holden*, James B Thodent and Frank M Raushel 679 Carbamoyl phosphate synthetase: a tunnel runs through it Hazel M Holden*, James B Thodent and Frank M Raushel The direct transfer of metabolites from one protein to another Biochemistry of carbamoyl phosphate in a biochemical pathway or between one active site and synthetase another within a single enzyme has been described as Carbamoyl phosphate synthetase, hereafter referred to as substrate channeling. The first structural visualization of such a CPS, plays a critical role in both arginine and pyrimidine phenomenon was provided by the X-ray crystallographic biosynthesis by providing an essential precursor, namely analysis of tryptophan synthase, in which a tunnel of carbamoyl phosphate. This remarkable enzyme has been approximately 25/~, in length was observed. The recently the focus of intense investigation for more than 30 years, determined three-dimensional structure of carbamoyl due, in part, to both its important metabolic role and the phosphate synthetase sets a new long distance record in that large number of substrates, products and effector mole- the three active sites are separated by nearly 1 O0 A. cules that bind to it. Addresses According to most biochemical data, CPS catalyzes the *tDepartment of Biochemistry, University of Wisconsin, Madison, production of carbamoyl phosphate from one molecule of W153706, USA bicarbonate, two molecules of MgZ+ATP and one molecule *e-mail: holden @enzyrne,wisc.edu re-mail: [email protected] of glutamine, as depicted in Scheme I below [3,4]. $Department of Chemistry, and Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA; e-maih [email protected] O O O Current Opinion in Structural Biology 1998, 8:679-685 II II II http://biomednet.com/elecreflO959440XO0800679 /ON HO O- MgADP HO/ \/i0 O-\ O- © Current Biology Ltd ISSN 0959-440X Bicarbonate Carboxyphosphate Abbreviations II ,o Pi NH3 4 == Gin CPS carbamoylphosphate synthetase Glu G PATase glutarnine phosphoribosylpyrophosphate amidotransferase O O 0 II II .l MgATP II /CN/~\ MgAD'P /C\ Introduction H2N O O- O- H2N O- The concept of substrate channeling, as described in [1], Carbamoylphosphate Carbamate was originally put forth to explain the manner by which reactive intermediates are transferred from one protein to another in a metabolic pathway or shuttled from one active site to another within a single enzyme. Although As can be seen, there are, at minimum, three reactive the biochemical evidence for substrate channeling was species--carboxyphosphate, with a half-life of approxi- substantial, the first direct structural observation of such mately 70 ms [5], ammonia and carbamate, with an a phenomenon was derived from the elegant X-ray crys- estimated half-life of 28 ms [6]. tallographic analysis of tryptophan synthase isolated from Salmonella typhimurium [2]. This investigation demon- As isolated from E. coli, the enzyme is composed of two strated that the two active sites, located on the ~ and 13 polypeptide chains, referred to as the large and small sub- subunits of the enzyme, are separated by a distance of units. The small subunit catalyzes the hydrolysis of approximately 25 A and are connected by a tunnel of the glutamine [7], while the large subunit is responsible for appropriate diameter to facilitate the diffusion of indole. the two phosphorylation events [8]. In addition, the large subunit provides the regions of the polypeptide chain As more complicated protein structures are solved to that are responsible for binding physiologically important increasingly higher resolution, it is becoming apparent monovalent cations and effector molecules, such as that substrate channels may, indeed, be quite common. ornithine, an activator, and UMP, an inhibitor [9,10]. So far, the long distance record for a channel between These inhibitors and activators effect the reaction pri- active sites has been set by carbamoyl phosphate syn- marily through the modulation of the Michaelis constant thetase from Escherichia coli, the focus of this review. In for MgZ+ATP [11,12]. this review, we present the structure of the enzyme and describe the channels that are essential for shuttling the Through the painstaking efforts of Lusty and co-workers reactive and unstable intermediates between three active [13,14], the genes encoding both the large and small sub- site regions. units were sequenced in the early 1980s; two key features 680 Catalysisand regulation Figure 1 Stereo view ribbon representation of the CPS (c~,l~)4 heterotetramer. The small subunits are displayed in magenta. The components of the large subunits are shown in green, yellow, blue and red, representing the regions defined by Met1 to Glu403, Va1404 to Ala553, Asn554 to Asn936 and Ser937 to Lys1073, respectively. were revealed from these studies. First, in the small sub- components, displayed in green and blue, respectively; an unit, the amino acid sequence of the C-terminal portion of oligomerization region, shown in yellow; and an allosteric the polypeptide chain was shown to be homologous to motif, depicted in red. These four regions of the large sub- sequences corresponding to the N-terminal domains of unit are delineated by Metl to Glu403, Va1404 to Ala553, trpG-type amidotransferases. Second, and more surprising, Asn554 to Ash936 and Ser937 to Lys1073, respectively. The in the large subunit, a homologous repeat sequence was molecular interactions between the small and large subunits found, such that residues Metl to Arg400 were 40% iden- of the CPS ~,13 heterodimer are quite extensive, with 35 tical to residues Ala553 to Leu933. direct hydrogen bonds between the two polypeptide chains [16°°]. Importantly, only residues in the carboxyphosphate Prior to the successful structural determination of the CPS synthetic component and oligomerization domain of the (a,l~) 4 tetramer in 1997 [15°°,16°'], it was unclear how as to large subunit contribute to the formation of this dimeric the enzyme was able to orchestrate the synthesis and sta- interface. There are no direct interactions between the bilization of three separate reaction intermediates. It was small subunit and the carbamoyl phosphate synthetic com- tacitly assumed that the active sites were situated near one ponent of the large subunit. In contrast to the another. Quite strikingly, however, the active sites are sep- subunit-subunit interface of the or,IS heterodimer, the num- arated by nearly 100 A, thereby implying some type of ber of interactions between one ~,13 heterodimer and substrate channeling, as discussed here. another within the complete tetramer is minimal [16°°]. The carbamoyl phosphate synthase In order to more fully appreciate the underlying architecture (~)4 tetramer of the ~,13 heterodimer, the following discussion will focus The overall three-dimensional motif of the CPS (~,~)4 on the individual parts. It should be kept in mind, however, tetramer, displayed in a ribbon representation in Figure 1, that all of these components are intimately associated with exhibits nearly exact 222 symmetry, with the rotational rela- each other and, ultimately, are dependent upon one another tionships between one ~,13 species and the other three being for the full enzymatic activity displayed by CPS. 179.9 °, 178.1 ° and 178.1 ° [15°°]. Color coded in magenta in Figure 1, the four small subunits of the (or,13)4 tetramer are The small subunit perched at the ends of the molecule. Each large subunit As can be seen in Figure 1, the polypeptide chain of the within the tetramer can be described in terms of four dis- small subunit folds into two distinct structural motifs. The tinct components: two units referred to as the N-terminal domain, formed by Leul to Leu153, contains carboxyphosphate and carbamoyl phosphate synthetic two 13-sheet layers oriented nearly perpendicular to one Carbamoyl phosphate synthetase Holden,Thoden and Raushel 681 Figure 2 Close-upstereo view of the smallsubunit activesite, with the boundglutamyl thioester F314/~::I~ F314~ G243 intermediateindicated by the filled black At~k ~ O G243 bonds. a~ ~ ~2~269S~241 ) ~ ~ - N240 . F350 H353N ~ E355 F350 Ha53N E35 o s O O O 0 0 Current Opinion in Structural Biology another. One of these sheets contains four parallel biochemical studies have suggested that the reaction 13 strands, whereas the other consists of four antiparallel mechanism proceeds through the formation of a cova- I~ strands. The C-terminal domain, delineated by Asn154 lently bound glutamyl thioester intermediate [19]. to Lys382, is dominated by a 10-stranded mixed [3 sheet Furthermore, the role of His353 in activating the cysteine and six (~ helices. This mixed ~ sheet is the only example for nucleophilic attack has been supported by site-direct- of this tertiary structural element in the entire ed mutagenesis experiments in which it was replaced CPS ~,~ heterodimer, as all other [3 sheets in the enzyme with an asparagine [20]. Recent X-ray crystallographic run either purely parallel or antiparallel. analyses of the H353N mutant have indeed confirmed the existence of the glutamyl thioester intermediate, Although three-dimensional structural searches have thus which was trapped in the active site [21"°]. As can be seen far failed to reveal any significant homology between the in Figure 2, it is absolutely clear that Or of Set47 and the N-terminal domain of the CPS small subunit and other backbone amide hydrogen of Gly241 are in an ideal loca- proteins of known structure, it is absolutely clear that the tion both to position the carbonyl carbon
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