Carbamoyl phosphate synthetase: a tunnel runs through it.

The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analysis of tryptophan synthase, in which a tunnel of approximately 25 A in length was observed. The recently determined three-dimensional structure of carbamoyl phosphate synthetase sets a new long distance record in that the three active sites are separated by nearly 100 A.

Carbamoyl phosphate synthetase: a crooked path from substrates to products.

The formation of carbamoyl phosphate is catalyzed by a single enzyme using glutamine, bicarbonate and two molecules of ATP via a reaction mechanism that requires a minimum of four consecutive reactions and three unstable intermediates. The recently determined X-ray crystal structure of carbamoyl phosphate synthetase has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein. It has been demonstrated that the amidotransferase domain within the small subunit of the enzyme from Escherichia coli hydrolyzes glutamine to ammonia via a thioester intermediate with Cys269. The ammonia migrates through the interior of the protein, where it reacts with carboxy phosphate to produce the carbamate intermediate. The carboxy phosphate intermediate is formed by the phosphorylation of bicarbonate by ATP at a site contained within the amino-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the carboxy-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate. The entire journey from substrate to product covers a distance of nearly 100 A.

High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol.

UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between beta-NADH and a 4-keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 A resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol. The model was refined to 1.8 A resolution with a final overall R-factor of 18.6%. This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDP-glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis. The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.

Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

Steroidal allylic fluorination using diethylaminosulfur trifluoride: a convenient method for the synthesis of 3 beta-acetoxy-7 alpha- and 7 beta-fluoroandrost-5-en-17-one.

This paper discusses our findings regarding fluorination of the diastereomeric 3 beta-acetoxy-7-hydroxyandrost-5-en-17-ones (3 and 4) at the allylic 7-hydroxyl group using diethylaminosulfur trifluoride under various experimental conditions. The reaction led to the formation of allylic 7 alpha- and 7 beta-fluoro derivatives, 6 and 7, contaminated with small amounts of 3 beta-acetoxy-5 alpha-fluoroandrost-6-en-17-one (8), the rearrangement product, and 3 beta-acetoxyandrosta-4,6-dien-17-one (9), the elimination product. However, synthesis of 3 beta-acetoxy-7 alpha-fluoroandrost-5-en-17-one (6) and 3 beta-acetoxy-7 beta-fluoroandrost-5-en-17-one (7) has been achieved in high isomeric purity by careful manipulation of the experimental conditions. Also included herein is a convenient chemical synthesis of pure 3 beta-acetoxy-7 alpha-hydroxyandrost-5-en-17-one (4) and 3 beta-acetoxy-7 beta-hydroxyandrost-5-en-17-one (3), the starting materials for the present fluorination reaction. The structure of a degradation product, 3 beta-acetoxy-5 alpha-hydroxyandrost-6-en-17-one (5), has been established by X-ray diffraction analysis to ascertain unambiguously its absolute configuration.

Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. Within recent years the enzyme from Escherichia coli has been studied extensively by both biochemical and X-ray crystallographic techniques. One of several key features in the catalytic mechanism of the enzyme involves the putative rotation of a 4'-ketopyranose intermediate within the active site region. The mode of binding of UDP-glucose to epimerase is well understood on the basis of previous high-resolution X-ray crystallographic investigations from this laboratory with an enzyme/NADH/UDP-glucose abortive complex. Attempts to prepare an enzyme/NADH/UDP-galactose abortive complex always failed, however, in that UDP-glucose rather than UDP-galactose was observed binding in the active site. In an effort to prepare an abortive complex with UDP-galactose, a site-directed mutant protein was constructed in which Ser 124 and Tyr 149, known to play critical roles in catalysis, were substituted with alanine and phenylalanine residues, respectively. With this double mutant it was possible to crystallize and solve the three-dimensional structures of reduced epimerase in the presence of UDP-glucose or UDP-galactose to high resolution. This study represents the first direct observation of UDP-galactose binding to epimerase and lends strong structural support for a catalytic mechanism in which there is free rotation of a 4'-ketopyranose intermediate within the active site cleft of the enzyme.

Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product.

Carbamoyl phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry, in which carbamoyl phosphate is produced from one molecule of bicarbonate, two molecules of Mg2+ ATP, and one molecule of either glutamine or ammonia. The carbamoyl phosphate so produced is utilized in the synthesis of arginine and pyrimidine nucleotides. It is also employed in the urea cycle in most terrestrial vertebrates. Due to its large size, its important metabolic role, and the fact that it is highly regulated, CPS has been the focus of intensive investigation for nearly 40 years. Numerous enzymological, biochemical, and biophysical studies by a variety of investigators have led to a quite detailed understanding of CPS. Perhaps one of the most significant advances on this topic within the last 2 years has been the successful X-ray crystallographic analysis of CPS from Escherichia coli. Quite unexpectedly, this structural investigation revealed that the three active sites on the protein are widely separated from one another. Furthermore, these active sites are connected by a molecular tunnel with a total length of approximately 100 A, suggesting that CPS utilizes this channel to facilitate the translocation of reaction intermediates from one site to another. In this review, we highlight the recent biochemical and X-ray crystallographic results that have led to a more complete understanding of this finely tuned instrument of catalysis.

Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli.

UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose through a mechanism involving the transient reduction of NAD+. Here we describe the X-ray structures for epimerase complexed with NADH/UDP, and NAD+/UDP, refined to 1.8 and 2.0 angstrom, respectively. The alpha-carbon positions for the two forms of the enzyme are superimposed with a root-mean-square deviation of 0.36 A. Overall, the models for the reduced and oxidized proteins are very similar except for the positions of several side chains including Phe 178 and Phe 218. The most striking difference between the oxidized and reduced enzymes is the conformation of the nicotinamide ring of the dinucleotide. In the reduced protein, the nicotinamide ring adopts the anti conformation while in the oxidized enzyme the syn conformation is observed. There are also significant structural differences in UDP binding between the oxidized and reduced forms of the protein which most likely explain the observation that uridine nucleotides bind more tightly to epimerase/NADH than to epimerase/NAD+. Both van der Waals and electrostatic interactions between epimerase and NAD+ are extensive with 35 contacts below 3.2 angstrom as would be expected for enzyme that binds the dinucleotide irreversibly. This is in sharp contrast to the patterns typically observed for the NAD+-dependent dehydrogenases which bind nucleotides in a reversible fashion. While it has been postulated that the active site of epimerase must contain a base, the only potential candidates within approximately 5 A of both the NAD+ and the UDP are Asp 31, Asp 58, and ASP 295. These amino acid residues, however, are intimately involved in nucleotide binding and most likely do not play a role in the actual catalytic mechanism. Thus it may be speculated that an amino acid residue, other than glutamate, aspartate, or histidine, may be functioning as the active site base.

Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase plays a critical role in sugar metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose. Originally, it was assumed that the enzyme contained a "traditional" catalytic base that served to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or UDP-galactose substrates during the course of the reaction. However, recent high-resolution X-ray crystallographic analyses of the protein from Escherichia coli have demonstrated the lack of an aspartate, a glutamate, or a histidine residue properly oriented within the active site cleft for serving such a functional role. Rather, the X-ray crystallographic investigation of the epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'- and 3'-hydroxyl groups of the sugar, respectively. To test the structural role of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant proteins, namely S124A, S124T, and S124V, were constructed and crystals of the S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T. NADH.UDP-glucose, and S124V.NADH.UDP-glucose complexes were grown. All of the crystals employed in this investigation belonged to the space group P3221 with the following unit cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric unit. X-ray data sets were collected to at least 2.15 A resolution, and each protein model was subsequently refined to an R value of lower than 19.0% for all measured X-ray data. The investigations described here demonstrate that the decreases in enzymatic activities observed for these mutant proteins are due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural perturbations. In addition, these structures demonstrate the importance of a hydroxyl group at position 124 in stabilizing the anti conformation of the nicotinamide ring as observed in the previous structural analysis of the epimerase.NADH. UDP complex.

Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism.

UDP-galactose 4-epimerase is one of three enzymes in the metabolic pathway that converts galactose into glucose1-phosphate. Specifically this enzyme catalyzes the interconversion of UDP-galactose and UDP-glucose. The molecular structure of the NADH/UDP-glucose abortive complex of the enzyme from Escherichia coli has been determined by X-ray diffraction analysis to a nominal resolution of 1.8 A and refined to an R-factor of 18.2% for all measurement X-ray data. The nicotinamide ring of the dinucleotide adopts the syn conformation in relationship to the ribose. Both the NADH and UDP-glucose are in the proper orientation for a B-side specific transfer from C4 of the sugar to C4 of the dinucleotide. Those residues implicated in glucose binding include Ser 124, tyr 149, Asn 179, Asn199, Arg 231, and Tyr 299. An amino acid sequence alignment of various prokaryotic and eukaryotic epimerases reveals a high degree of conservation with respect to those residues involved in both NADH and substrate binding. The nonstereospecificity displayed by epimerase was originally thought to occur through a simple rotation about the bond between the glycosyl C1 oxygen of the 4-ketose intermediate and the beta-phosphorous of the UDP moiety, thereby allowing the opposite side of the sugar to face the NADH. The present structure reveals that additional rotations about the phosphate backbone of UDP are necessary. Furthermore, the abortive complex model described here suggests that Ser 124 and Tyr 149 are likely to play important roles in the catalytic mechanism of the enzyme.

The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.

The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechanism displayed by this class of enzymes is provided by carbamoyl phosphate synthetase (CPS). Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia. Kinetic investigations have led to a proposed chemical mechanism for this enzyme that requires carboxy phosphate, ammonia, and carbamate as kinetically competent reaction intermediates. The three-dimensional X-ray crystal structure of CPS has localized the positions of three active sites. The nucleotide binding site within the N-terminal half of the large subunit is required for the phosphorylation of bicarbonate and subsequent formation of carbamate. The nucleotide binding site within the C-terminal domain of the large subunit catalyzes the phosphorylation of carbamate to the final product, carbamoyl phosphate. The three active sites within the heterodimeric protein are separated from one another by about 45 A. The ammonia produced within the active site of the small subunit is the substrate for reaction with the carboxy phosphate intermediate that is formed in the active site found within the N-terminal half of the large subunit of CPS. Since the ammonia does not dissociate from the protein prior to its reaction with carboxy phosphate, this intermediate must therefore diffuse through a molecular tunnel that connects these two sites with one another. Similarly, the carbamate intermediate, initially formed at the active site within the N-terminal half of the large subunit, is the substrate for phosphorylation by the ATP bound to the active site located in the C-terminal half of the large subunit. A molecular passageway has been identified by crystallographic methods that apparently facilitates diffusion between these two active sites within the large subunit of CPS. Synchronization of the chemical transformations is controlled by structural perturbations among the three active sites. Molecular tunnels between distant active sites have also been identified in tryptophan synthase and glutamine phosphoribosyl pyrophosphate amidotransferase and are likely architectural features in an expanding list of enzymes.

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.

Movement of the biotin carboxylase B-domain as a result of ATP binding.

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-A resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the three-dimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-A resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45(o) of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.

Crystallization and preliminary X-ray crystallographic analysis of carbamoyl phosphate synthetase from Escherichia coli.

Carbamoyl Phosphate synthetase catalyzes the formation of carbamoyl phosphate, a high-energy intermediate used in several biosynthetic pathways. The enzyme from Escherichia coli has been crystallized at pH 8 in the presence of L-ornithine, MnCl(2) and ADP, using PEG 8000 in combination with NEt(4)Cl and KCl. The crystals (apparently) belong to the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions of a = 154.4, b = 166.5 and c = 338.7 A. The crystals are relatively sensitive to radiation damage, but show diffraction to beyond 2.8 A resolution. A low-resolution (3.5 A) native data set has been recorded and conditions for flash cooling the crystal have been established.

Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product.

Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.

Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily.

Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway.

Galactokinase: structure, function and role in type II galactosemia.

The conversion of beta- D-galactose to glucose 1-phosphate is accomplished by the action of four enzymes that constitute the Leloir pathway. Galactokinase catalyzes the second step in this pathway, namely the conversion of alpha- D-galactose to galactose 1-phosphate. The enzyme has attracted significant research attention because of its important metabolic role, the fact that defects in the human enzyme can result in the diseased state referred to as galactosemia, and most recently for its utilization via 'directed evolution' to create new natural and unnatural sugar 1-phosphates. Additionally, galactokinase-like molecules have been shown to act as sensors for the intracellular concentration of galactose and, under suitable conditions, to function as transcriptional regulators. This review focuses on the recent X-ray crystallographic analyses of galactokinase and places the molecular architecture of this protein in context with the extensive biochemical data that have accumulated over the last 40 years regarding this fascinating small molecule kinase.

Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding.

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.

The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution.

Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxyphosphate and carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.