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Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit

2004; Springer Nature; Volume: 23; Issue: 18 Linguagem: Inglês

10.1038/sj.emboj.7600373

1460-2075

Pamela R. Hall, Run Zheng, Lizamma Antony, Marianne Pusztai‐Carey, Paul Carey, Vivien C. Yee,

Enzyme Structure and Function

Article26 August 2004free access Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit Pamela R Hall Pamela R Hall Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Run Zheng Run Zheng Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Lizamma Antony Lizamma Antony Department of Oncology, Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Marianne Pusztai-Carey Marianne Pusztai-Carey Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Paul R Carey Paul R Carey Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Vivien C Yee Corresponding Author Vivien C Yee Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Pamela R Hall Pamela R Hall Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Run Zheng Run Zheng Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Lizamma Antony Lizamma Antony Department of Oncology, Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Marianne Pusztai-Carey Marianne Pusztai-Carey Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Paul R Carey Paul R Carey Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Vivien C Yee Corresponding Author Vivien C Yee Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Author Information Pamela R Hall1,2, Run Zheng2, Lizamma Antony3, Marianne Pusztai-Carey2, Paul R Carey2 and Vivien C Yee 1,2 1Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA 2Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA 3Department of Oncology, Johns Hopkins University, Baltimore, MD, USA *Corresponding author. Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue RT500, Cleveland, OH 44106, USA. Tel.: +1 216 368 1184; Fax: +1 216 368 3419; E-mail: [email protected] The EMBO Journal (2004)23:3621-3631https://doi.org/10.1038/sj.emboj.7600373 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transcarboxylase is a 1.2 million Dalton (Da) multienzyme complex from Propionibacterium shermanii that couples two carboxylation reactions, transferring CO2− from methylmalonyl-CoA to pyruvate to yield propionyl-CoA and oxaloacetate. Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in free form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2-ketobutyrate. The structure reveals a dimer of β8α8 barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex in which the product's carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S-based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites. Introduction Nature has selected biotin as a cofactor to be a carrier for carbon dioxide (CO2). In the form of carboxybiotin, CO2 is less reactive and at the site of delivery the transferred carboxyl group can be released as CO2 or directly to an acceptor nucleophile (Knowles, 1989). Biotin-dependent carboxylases function through two partial reactions and fall into three classes (Wood and Barden, 1977). Class I enzymes, which include all eukaryotic biotin enzymes, first use ATP, Mg(II), and bicarbonate to form carboxybiotin, and then transfer the carboxyl group to an acceptor molecule such as pyruvate or acetyl-CoA. Class II enzymes couple decarboxylation of β-keto acids and their thioesters with sodium transport in anaerobic prokaryotes. Transcarboxylase (TC) from Propionibacterium shermanii is the only member of Class III, and couples two carboxylation reactions. To date, only four mammalian biotin-dependent carboxylases have been identified: acetyl-CoA carboxylase (ACC), methylcrotonoyl-CoA carboxylase (MCC), propionyl-CoA carboxylase (PCC), and pyruvate carboxylase (PC) (Moss and Lane, 1971; Samols et al, 1988). These Class I carboxylases play central roles in such metabolic pathways as oxidation of odd-chain fatty acids, catabolism of branched amino acids, fatty acid synthesis, and gluconeogenesis. As the first enzyme in the gluconeogenic pathway, PC catalyzes the ATP-driven conversion of pyruvate to oxaloacetate using HCO3− and Mg2+ (Wexler et al, 1994). Deficiencies of PC result in lactic acidemia and may present as developmental delay, severe mental retardation or death by 3 months of age (Robinson 2001). TC has historically served as a model system for biotin-dependent carboxylases because its subunits share significant sequence homology with the important related human enzymes, are easily isolated, and form stable substrate complexes in the absence of the other subunits. TC is a 1.2 million Da multienzyme complex containing 30 polypeptide chains: a catalytic 336 kDa 12S hexameric core, six catalytic 116 kDa 5S dimers, and twelve 12 kDa 1.3S biotinylated linkers (Wood and Zwolinski, 1976) (Figure 1A). The overall TC transcarboxylation reaction consists of two half reactions (Wood and Kumar, 1985). In the first half reaction, 12S transfers CO2− from MMCoA to biotin on 1.3S. 5S transfers the CO2− from the 1.3S biotin to pyruvate in the second half reaction (Figure 1B). TC's organization, in which intermediates are shuttled between different catalytic subunits by a flexible carrier, places it in the general group of multienzyme complexes such as pyruvate dehydrogenase (Coppel et al, 1988; Ho and Patel, 1990; Koike et al, 1990) and glycine decarboxylase (Kume et al, 1991). High-resolution structures of their individual subunits may provide some insight into the mechanism and holo enzyme organization as ambitious efforts continue to crystallize complete multienzyme complexes. Figure 1.The TC multienzyme complex. (A) EM-based model (adapted from Wrigley et al, 1977). (B) The two half reactions catalyzed by the 12S and 5S subunits, and the overall full reaction. Download figure Download PowerPoint Crystal structures of TC subunits are important for a mechanistic insight into mammalian Class I enzymes, which have been difficult to express in large quantities and to crystallize. We have previously described the 12S crystal structure and its relevance to PCC (Hall et al, 2003). The other large TC subunit, 5S, is functionally and sequentially homologous (27% identity) to the C-terminal carboxyltransferase region of human pyruvate carboxylase (EC 6.4.1.1) (Thorton et al, 1993; Wexler et al, 1994). No high-resolution three-dimensional structure is currently available for a mammalian PC; thus, elucidation of the 5S crystal structure will provide a mechanistic insight into both TC and related carboxyltransferases such as PC. We have solved the crystal structure of TC wild-type 5S as free protein and as complexes bound to its substrate pyruvate, product oxaloacetate or competitive inhibitor 2-ketobutyrate. These structures reveal the protein fold, dimer organization, and active site features important for substrate binding and catalysis. The 5S structure serves as a scaffold for the homology modeling of the human PC carboxyltransferase domain, which supports the expectation of conserved fold, active site, and mechanism. We have designed two 5S mutants which mimic PC disease mutations. They have the same level of reduced enzymatic activity as their PC counterparts, explained by their crystal structures, thus reinforcing the value of TC as a model for mammalian biotin-dependent carboxylases. Results and discussion We have determined the crystal structures of TC 5S in free form and bound to pyruvate (5S-pyr), oxaloacetate (5S-oxal), and 2-ketobutyrate (5S-2keto), as well as of two single-site mutants (5S-A59T, 5S-M186I) (Table I). The free 5S structure was solved first, by selenomethionine MAD-phasing methods, and refined to 1.9 Å resolution. Its protein coordinates were then used to solve the other 5S structures at either 2.0 Å (5S-pyr, 5S-2keto), 2.5 Å (5S-oxal, 5S-A59T), or 2.8 Å (5S-M186I) resolution. All six 5S structures are very similar: superpositions of the complex and mutant structures on free 5S result in an r.m.s. fit of 0.2–0.3 Å for 471 equivalent Cα atoms. Unless otherwise noted, the highest resolution structure, of free 5S, is described in the following sections. Table 1. Data collection, phasing, and refinement statistics MAD phasing SeMet-5S SeMet-5S SeMet-5S Data processing Source X25 X25 X25 Wavelength (Å) 1.0734 0.9803 0.9808 Unit cell (Å3) 96.3 96.3 96.3 147.1 147.2 147.2 79.1 79.1 79.1 Resolution (Å) 50–2.0 (2.07–2.0) 50–2.0 (2.07–2.0) 50–2.0 (2.07–2.0) 〈I〉/〈σ(I)〉 19.5 (5.3) 17.1 (3.7) 16.4 (3.4) Rmerge (%) 5.3 (14.9) 5.6 (18.9) 4.5 (17.7) Completeness (%) 93.3 (83.2) 90.2 (77.4) 66.8 (54.3) Phasing Resolution (Å) 20–2.5 Number of sites 23 Mean f.o.m. 0.66 Structures 5S 5S-pyr 5S-oxal 5S-2keto 5S-M186I 5S-A59T Data collection Source X25 Rigaku Rigaku Rigaku Bruker Rigaku Wavelength (Å) 0.9803 1.5413 1.5413 1.5413 1.5413 1.5413 Unit cell (Å3) 96.5 96.0 95.9 96.2 95.7 96.3 145.9 145.7 145.5 146.1 147.0 146.3 79.0 78.7 78.2 78.7 78.8 78.7 Resolution (Å)a 50–1.9 30–2.0 30–2.5 30–2.0 30–2.8 30–2.5 (1.97–1.9) (2.07–2.0) (2.59–2.5) (2.07–2.0) (2.93–2.8) (2.59–2.5) 〈I〉/〈σ(I)〉 19.5 (6.5) 16.3 (4.3) 22.8 (7.2) 22.0 (4.5) 5.6 (2.7) 22.0 (6.9) Rmerge (%) 7.0 (16.4) 8.7 (21.8) 6.6 (15.3) 8.4 (23.4) 10.0 (22.4) 6.9 (17.7) Completeness (%) 95.7 (87.0) 92.8 (81.0) 93.5 (86.3) 85.3 (71.8) 99.3 (99.5) 94.8 (87.5) Refinement Rwork, Rfree 16.24, 19.07 16.42, 19.63 15.25, 22.59 17.31, 20.45 20.78,26.92 20.52, 25.00 r.m.s.d. bond lengths (Å) 0.0072 0.0072 0.0073 0.0071 0.0078 0.0075 Bond angles (deg) 1.28 1.22 1.35 1.27 1.33 1.31 Number of atoms: protein 3664 3677 3661 3672 3657 3666 Ions, substrate 1, 0 1, 6 1, 9 1, 7 1, 0 1, 0 Solvent 372 415 188 260 31 1 B-factor, mean (Å)b Protein 25.2 31.9 31.2 34.9 34.3 19.6 Bound ligand 20.7 33.0 26.2 Protein within 8 Å of ligand 21.6 18.9 24.1 Co+2 ion 12.0 28.6 21.1 24.2 35.2 29.9 Co+2 ligands 11.5 23.6 20.8 16.1 28.6 16.2 Solvent 32.4 37.3 29.3 35.4 20.3 13.2 r.m.s.d., bonded 2.4 1.9 2.2 2.1 2.2 0.7 r.m.s.d., angled 3.1 2.6 3.3 2.9 3.4 1.3 Ramachandran (%) most fav., disallow. 92.3, 0.0 92.0, 0.0 88.4, 0.0 92.0, 0.0 86.4, 0.2b 89.1, 0.0 Values in parentheses are for the highest resolution shell. aSame resolution limits were used for data processing and refinement. bLeu97 refines into a disallowed region in one structure; the corresponding density and structural environment do not explain this observation and this residue is in the allowed region in all other structures. Overall structure Native 5S monomer has 505 residues; the recombinant 5S which was crystallized contains extended termini due to cloning artifacts and the C-terminal His6 tag. The asymmetric unit contains a monomer with overall dimensions ∼50 Å × 60 Å × 56 Å. No density is observed for N- and C-terminal residues, which are presumed to be flexible. The final refined structure includes protein residues 3–474, one cobalt ion, and 372 water molecules (Table I). 5S contains the canonical TIM-barrel fold (Banner et al, 1975), which consists of a core β8α8 motif with the eight parallel β strands forming an enclosed barrel surrounded by eight α helices (Figure 2). In addition, 5S has an N-terminal β strand (β1), two short α helices between β1 and α1 and between β2 and α2 (termed α1a and α2a, respectively), and a large C-terminal extension starting at residue Gly265 that contains two β strands and nine α helices. The C-terminal extension forms a funnel leading to the active site at the C-termini of the parallel β strands. Figure 2.Stereoview ribbon diagram of the 5S dimer. The bottom monomer is colored with its core eight-stranded β-barrel in yellow, the surrounding α helices in cyan, the C-terminal extension in purple, and the active site cobalt ion shown as a pink sphere. The second (top) monomer is generated from the first by a 90° rotation about the vertical axis and a 180° rotation about an axis perpendicular to the page, and is shown in paler colors. The N- and C-termini for the two monomers are labeled. Download figure Download PowerPoint Structural similarity to other TIM-barrel proteins Not surprisingly, a DALI search (Holm and Sander, 1998) matched 5S with a large number of proteins with similar folds (Table II). The TIM-barrel domain is estimated to be present in ∼10% of known enzyme structures (Farber and Petsko, 1990) and is utilized by at least 15 different enzyme families, with the active site always located at the C-termini of the β strands (Wierenga, 2001; Nagano et al, 2002). The top DALI match is 4-hydroxy-2-oxovalerate aldolase, which has, in addition to the TIM barrel, a C-terminal extension (Gly250–His340) corresponding to the first half of the 5S C-terminal extension (Gly265–Gly366). The remaining DALI matches share only the TIM barrel with 5S. Of these, seven structures are of particular interest since they are (de)carboxylases, although none are biotin-dependent. The active sites of these seven TIM-barrel (de)carboxylase structures show little conservation with 5S, with the exception of ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco), whose active site structure will be discussed in more detail later. Table 2. TIM-barrel proteins structurally similar to 5S Protein DALI Z-score Sequence identity (%) r.m.s.d. (Å), # equivalent Cα Reference 4-hydroxy-2-oxovalerate aldolase 28.6 15 3.4, 308 Manjasetty et al, 2003 Orotidine monophosphate decarboxylase 14.9 10 3.1, 203 Wu et al, 2000 3-keto-L-gulonate 6-phosphate decarboxylase 13.8 11 3.0, 194 Wise et al, 2002 Ribulose-1,5bisphosphate carboxylase 9.2 7 3.9, 193 Lundqvist and Schneider, 1991 Uroporphyrinogen decarboxylase 9.2 7 3.9, 188 Whitby et al, 1998 Ornithine decarboxylase 7.3 4 6.2, 188 Kern et al, 1999 Diaminopimelate decarboxylase 7.2 6 4.7, 196 Gokulan et al, 2003 Phosphoenolpyruvate carboxylase 7.0 7 6.1, 246 Kai et al, 1999 Selected from a total of 247 hits with Z-scores >2.0. Dimer organization The 5S subunit is a homodimer of 116 kDa that is generated by applying crystallographic symmetry to the monomer in the asymmetric unit. Residues from both the TIM-barrel domain and C-terminal extension form the extensive dimerization interface, which buries an average of 3921 Å2 total accessible surface area and is 56% hydrophobic (Figure 2). The dimerization interface also has a high shape complementarity index of 0.63 (Lawrence and Colman, 1993). Eight hydrogen bonds align the two N-termini to form an antiparallel intermolecular β-sheet. To confirm that the crystallized dimer is also observed in solution, we prepared the single-site mutants Lys229Glu and Glu232Lys to remove the key Lys229–Glu232 salt bridge at the dimer interface, and found that the mutants were a mixture of monomer and dimer in solution, while wild-type 5S and two control mutants altering other crystal contacts are essentially completely dimeric (data not shown). Cobalt-binding site 5S is a cobalt-dependent metalloenzyme (Northrop and Wood, 1969). The presence of a bound metal ion was shown by anomalous difference density, and its identification as cobalt was confirmed by ICP-MS. In the 5S active site, there is a single cobalt ion octahedrally coordinated by the side chains of His215, His217, and Asp23, a water molecule (WatA), and the CO2− from the carbamylated Lys184 (denoted LysC184; Figure 3A). All cobalt ligands are conserved in PC, as expected if the two proteins have a conserved mechanism (Figure 4A). The tight coordination sphere of the cobalt ion, with an average ligand distance of 2.15 Å, is consistent with reports that the 5S metal can be removed only under protein denaturing conditions (Ahmad et al, 1972). The cobalt ion is found at the base of a deep active site cavity; its His215, His217, and Asp23 ligands are in mostly well-packed environments with little freedom for substantial movement, while in contrast LysC184 and WatA are relatively solvent-accessible and have space to move in and out of the cobalt coordination sphere. Figure 3.5S active site. The cobalt ion (pink sphere), its water ligands, and side chains of residues which interact with either the cobalt ion or substrate (ball-and-stick) are shown. Potential interactions involving the metal ion and active site ligands are represented by pink and gray dashed lines, respectively. (A) Free 5S: 50–1.9 Å resolution simulated annealing ∣Fo∣–∣Fc∣ omit density contoured at 7σ shows a carbamylated LysC184 that coordinates the cobalt ion. (B) 5S-oxal: 30–2.5 Å resolution simulated annealing ∣Fo∣–∣Fc∣ omit density contoured at 4σ shows bound oxaloacetate interacting with the cobalt ion and a noncarbamylated Lys184 interacting with Cys154. (C) 5S-pyr: 30–2.0 Å resolution simulated annealing ∣Fo∣–∣Fc∣ omit density contoured at 4σ shows bound pyruvate (refined at half occupancy) and two conformations for Lys184/LysC184 (both refined at half occupancy). (D) 5S-2keto: 30–2.0 Å resolution simulated annealing ∣Fo∣–∣Fc∣ omit density contoured at 3σ shows bound 2-ketobutyrate (refined at half occupancy) and two conformations for Lys184/LysC184 (both refined at half occupancy). Download figure Download PowerPoint Figure 4.5S homology with PC. (A) Structure-based sequence alignment of 5S with the carboxyltransferase region of human PC (SwissProt accession number P11498), with 5S secondary structure elements colored similarly to their representation in Figure 2. Conserved residues (cyan), sites of PC missense disease mutations (*), the carbamylated Lys184 and the known/potential Cys partner residues (underlined), and the aligned putative pyruvate binding and metal-binding motifs (boxed) are highlighted. (B) Space-filling representation of 5S monomer with view into the active site. Residues conserved in PC are in blue, cobalt ion in pink, and the remaining residues colored by secondary structure and domain location as in Figure 2. (C) As in (B), but rotated 90° about the vertical axis to show the dimerization surface with its few conserved residues. Download figure Download PowerPoint Free 5S active site The most intriguing feature of the free 5S active site is the unexpected carbamylation of residue LysC184 (Figure 3A). In addition to coordinating the cobalt ion with distances of 2.2 and 2.3 Å, the CO2− modification of the LysC184 side chain also forms hydrogen bonds with His215, Arg22, Asp23, and two waters, WatA and WatB. Lys184, its amino-acid hydrogen-bonding partners, and nearly all the solvent-exposed active site residues are conserved in PC (Figure 4B). Some of these are of particular interest and will be discussed in more detail later: Gln26, which is located in helix α1a along with Arg22 and Asp23; Ala59 on α2a, Cys154 on strand β5, and Met186 on β6. 5S-oxal complex In the 5S-oxal structure, clear density is observed for the product oxaloacetate bound in the 5S active site (Figure 3B). Oxaloacetate was refined at three-quarter occupancy to minimize residual difference electron density; as a result, the oxaloacetate average atomic temperature factor of 33 Å3 is higher than that of 19 Å3 for the surrounding protein atoms (Table I). Attempts were made to fit oxaloacetate in two opposing orientations; one clearly fit the electron density better. In the correct orientation, the 'transferred' CO2− of oxaloacetate coordinates the active site cobalt, with the carboxylate oxygen atoms 1.6 and 3.0 Å from the metal ion. Oxaloacetate thus effectively replaces LysC184 as a cobalt ligand. With the two crystal structures superimposed, the 'transferred' oxaloacetate CO2− carbon in 5S-oxal is nearly coincident with (only 1.0 Å from) the corresponding carbon atom of LysC184 in free 5S, and is 2.7 Å from the cobalt ion. In the middle of oxaloacetate, the carbonyl oxygen forms hydrogen bonds with Arg22 and Gln26; the adjacent carboxylate group is partially solvent-exposed and not involved in any electrostatic interactions. There are two additional pronounced differences between the 5S-oxal and free 5S active sites. First, in 5S-oxal, Lys184 is not carbamylated as in free 5S; it is pointed away from the cobalt and its amine forms a 2.8 Å hydrogen bond with the sulfhydryl of Cys154. Second, there is no WatA equivalent in 5S-oxal and, as a result, the cobalt ion in the oxaloacetate complex has an incomplete coordination sphere. 5S-pyr complex The isolated 5S subunit does not have measurable catalytic activity; it is only active in the presence of the other TC subunits (Xie et al, 1993). This made it possible to crystallize the complex of 5S bound to its pyruvate substrate. In the 5S-pyr structure, pyruvate is not directly bound to the cobalt—the closest approach is its C3 methyl carbon, the target for carboxylation during the 5S reaction, which is 5.2 Å from the metal ion (Figure 3C). This distance, along with cobalt to pyruvate carbonyl carbon and carboxylate carbon distances of 5.3 and 6.8 Å, are near those reported earlier (6.3, 5.0, and 6.3 Å, respectively) using EPR and NMR (Fung et al, 1974). In effect, pyruvate is bound in the same position and orientation as oxaloacetate in 5S-oxal, such that there is space between the pyruvate and cobalt for the insertion of the CO2− group to take place during the carboxyltransferase reaction; the pyruvate C3 atom is 1.4 Å from the equivalent C2 of oxaloacetate when the 5S-pyr and 5S-oxal structures are superimposed. As also seen for the oxaloacetate carbonyl, the pyruvate carbonyl group forms hydrogen bonds with Arg22 and Gln26. New ligand interactions include hydrogen bonds between the pyruvate carbonyl oxygen and WatA, a solvent molecule observed in free 5S but not in 5S-oxal, and between the pyruvate carboxylate and Gln26. The electron density indicates two conformational and chemical states for Lys184, corresponding to half occupancies of noncarbamylated Lys184 as observed in 5S-oxal and carbamylated LysC184 as observed in free 5S. In 5S-pyr, the half-occupied LysC184 has its CO2− carbon 3.7 Å from the C3 atom of pyruvate. The half-occupied noncarbamylated Lys184 has shifted 3.7 Å to form a 2.6 Å long hydrogen bond with Cys154. The short pyruvate C3 to LysC184 distance can be explained by partial pyruvate occupancy; pyruvate was refined at half occupancy to minimize the residual difference electron density. As a result, the average atomic temperature factors for the ligand and surrounding protein atoms are both around 21 Å3 (Table I). 5S-2keto complex The competitive inhibitor 2-ketobutyrate blocks transcarboxylation due to its ethyl group in place of the methyl moiety in pyruvate. In the 5S-2keto structure, 2-ketobutyrate is positioned in the 5S active site similarly to pyruvate and oxaloacetate in 5S-pyr and 5S-oxal, respectively. The 2-ketobutyrate inhibitory carbon C4 is 4.6 Å from the cobalt, and the blocked transcarboxylation 'target' carbon C3 is 5.7 Å from the metal ion (Figure 3D). Thus, it is clear how 2-ketobutyrate is chemically and sterically incapable of functioning as substrate. 2-ketobutyrate is bound in 5S-2keto in essentially the same manner as pyruvate is bound in 5S-pyr (Figure 5). 5S-2keto also has half occupancies of noncarbamylated Lys184 forming a 2.8 Å hydrogen bond to Cys154 and of carbamylated LysC184 coordinated to the cobalt ion. As speculated for 5S-pyr, in 5S-2keto the short 3.2 Å distance between the 2-ketobutyrate C3 atom and the CO2− carbon of LysC184 can be explained by partial 2-ketobutyrate occupancy. This interpretation is consistent with the refinement of 2-ketobutyrate at half occupancy to minimize residual difference electron density, with the result that the average atomic Bs for the ligand and surrounding protein atoms are both around 25 Å3 (Table I). Figure 5.Active sites of 5S complexes. The free 5S active site is shown with its LysC184 (ball-and-stick with gray carbon atoms and bonds), cobalt ion (pink sphere), water ligands (red spheres), and side chains of residues which interact with either the cobalt ion or bound ligand. Superimposed are oxaloacetate (yellow), pyruvate (pale pink), and 2-ketobutyrate (cyan) ligands from their respective complexes. Download figure Download PowerPoint Homology modeling of pyruvate carboxylase The gluconeogenic enzyme PC is a homotetramer in which each 125 kDa subunit has a covalently bound biotin and binding sites for acetyl CoA, ATP, HCO3−, and pyruvate (Bardin et al, 1975; Wallace and Easterbrook-Smith, 1985). PC and TC 5S share functional homology and the requirement for a bivalent metal ion (Jitrapakdee and Wallace, 1999). The 5S sequence is 27% identical to that of the PC carboxyltransferase region, with the two most conserved motifs predicted to be the pyruvate-binding site (Samols et al, 1988) and metal-binding site (HXHXH) (Jitrapakdee and Wallace, 1999) (Figure 4A). In 5S, the His215 and His217 cobalt ligands correspond to the second and third histidine residues in the putative PC metal-binding site. In contrast, 5S residues Glu54–Leu76 correspond to the putative PC pyruvate-binding site, but do not interact with any of the ligands in our 5S crystal structures. We have constructed a 5S-based homology model of the carboxyltransferase domain of human PC. Most of the conserved residues are in the TIM barrel, with many of these solvent-inaccessible and likely to be structurally important. Of the solvent-exposed conserved residues, most are in the active site cavity, including Lys184 that is carbamylated in 5S (Lys741 in PC). Nearly all these solvent-accessible active site cavity residues are conserved, including all the amino acids involved in binding cobalt, oxaloacetate, pyruvate, or 2-ketobutyrate (Figure 4B). This observation is consistent with 5S and the PC carboxyltransferase domain having similar substrate-binding sites and catalytic mechanisms. In contrast, very few of the residues at the 5S dimer interface are conserved in PC (Figure 4C), which is not surprising since the TC multienzyme and homotetrameric PC have very different oligomeric organizations. Structural insight into PC disease mutations PC deficiency is an autosomal recessive disorder (Atkin et al, 1979) that presents in a number of forms ranging from mild to severe lactic acidemia (Robinson 2001). Of the four single-site missense mutations identified in PC deficiency, two occur in the carboxyltransferase region of PC. These PC mutations, A610T and M743I, alter residues corresponding to the conserved 5S amino acids A59 and M186 (Figure 4A). To confirm that 5S is a useful model to study PC disease mutants, we prepared the 5S mutants A59T and M186I that are homologous to the two PC disease mutations. Activity assays of 5S-A59T and 5S-M186I showed low relative specific activities ranging up to 3.6% of wild type (Table III). These values are similar to published activity measurements of the two PC disease mutants ranging from 0 to 17% that of control values (de Vivo et al, 1977; Atkin et al, 1979; Murphy et al, 1981; Robinson et al, 1984; Robinson, 2001). Table 3. Enzymatic activity of 5S mutants Relative specific activity (s.d.)a %Wild-type activity Wild type 1.93 (0.38) 100 Ala59Thr 0.07 (0.04) 3.6 Met186Ile 0.04 (0.03) 2.2 Lys184Ala −0.02 (0.13) 0 Lys184Glu −0.13 (0.21) 0 Cys154Ala 0.96 (0.51) 49.6 Statistics are based on experiments performed in triplicate on two separate protein preparations, and are representative of experiments performed on multiple 5S preparations. To obtain a structural view of the molecular consequence of the two PC disease mutants, we have determined the crystal structures of 5S-A59T and 5S-M186I (Table I). Both mutations alter exposed active site residues and do not change the local protein fold (Figure 6). In addition, as observed in wild-type free 5S, LysC184 is observed only in its carbamylated, cobalt-coordinating conformation in the two unliganded 5S mutant structures. In the wild-type 5S crystal structures, the Met186 side chain is found in two conformations: it packs against LysC184 in free