Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes*

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 Å, respectively. Comparison of these β/α-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg2+ coordination and positioning of the flexible loop containing the conserved HMGCL “signature” sequence. In the R41M-Mg2+-substrate ternary complex, loop residue Cys266 (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg2+-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg2+ liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His233 and His235 imidazoles, other Mg2+ ligands are the Asp42 carboxyl oxygen and an ordered water molecule. This water, positioned between Asp42 and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg41 with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg41 mutation on reaction product enolization and explains why human Arg41 mutations cause drastic enzyme deficiency.

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 Å , respectively. Comparison of these ␤/␣-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg 2؉ coordination and positioning of the flexible loop containing the conserved HMGCL "signature" sequence. In the R41M-Mg 2؉substrate ternary complex, loop residue Cys 266 (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg 2؉ -3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg 2؉ liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His 233 and His 235 imidazoles, other Mg 2؉ ligands are the Asp 42 carboxyl oxygen and an ordered water molecule. This water, positioned between Asp 42 and the C3 hydroxyl of bound substrate/ inhibitor, may function as a proton shuttle. The observed interaction of Arg 41 with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg 41 mutation on reaction product enolization and explains why human Arg 41 mutations cause drastic enzyme deficiency.
HMG-CoA lyase (HMGCL 4 ; EC 4.1.3.4) catalyzes a cationdependent cleavage of substrate into acetyl-CoA and acetoacetate (Scheme 1) (1). This reaction is a key step in ketogenesis, the products of which support anaplerotic metabolism in bacteria (2) and energy production in nonhepatic animal tissues (3). Ketogenesis is particularly important to human metabolism during the prenatal period and during fasting or starvation. In accordance with these physiological roles, it is not surprising that gene knock-out in mice results in embryonic lethality (4). The physiological importance of the enzyme in humans is underscored by the observation that mutations that diminish HMGCL activity correlate with inherited metabolic disease that can be lethal if uncontrolled (5).
A variety of human mutations, including many point mutations in protein-coding exons of the gene, have been documented (6). A computational modeling approach was used to explain the molecular basis for some mutations linked to inherited disease (7). This led to the prediction that HMGCL adopts a ␤/␣-barrel fold and a proposal that the acyl-S-pantetheine moiety of the bound substrate passes through the barrel lumen. Initial structural work on human HMGCL liganded to cation and hydroxyglutarate (8) demonstrated that the folding prediction was reasonable but that the substrate binding proposal was unlikely to be correct. The positions of bound cation and hydroxyglutarate (from hydrolyzed hydroxyglutaryl-CoA) indicated the catalytic site to be positioned at the C-terminal end of the barrel, but the absence of a full acyl-CoA molecule in the experimentally determined structure limited detailed insight into the substrate-binding site.
To more fully address questions regarding the conformation of bound substrate, activator cation liganding, details concerning reaction chemistry and specificity, as well as the molecular basis for certain inherited HMGCL deficiencies, new structural information on enzyme bound to an intact acyl-CoA molecule is required. To remedy this need, complexes of the WT enzyme with the competitive inhibitor 3-hydroxyglutaryl-CoA and also of catalytically deficient R41M enzyme with the authentic substrate HMG-CoA have been supplemented with the activator cation Mg 2ϩ , and crystallization of the desired ternary complexes has been accomplished. Diffraction quality crystals have been produced, supporting three-dimensional structural determinations for ternary complexes of enzyme, cation, and either the acyl-CoA substrate or inhibitory analog. These findings are now described and interpreted to address the questions outlined above.

EXPERIMENTAL PROCEDURES
Expression and Isolation of HMGCL Proteins-The WT and R41M mutant HMGCL proteins were expressed using plasmid pTrc-HL-1 and purified using the protocol developed for the WT enzyme (9). Cloning, expression, and purification of the R41M mutant were carried out as described previously (10). For investigation of the functional consequences of Lys 48 mutations, N-terminally His 6 -tagged proteins were produced by ligation of the NcoI/BamHI coding fragment (derived from pTrc-HL-1) into comparably restricted pET30b. The QuikChange mutagenesis protocol (Stratagene) was used to introduce mutations encoding K48N and K48Q. After DNA sequencing confirmed the desired mutations and the absence of artifacts in the coding sequence, the plasmids were used for protein expression. Active enzyme was expressed, and the efficient isolation of homogeneous N-terminally His-tagged proteins was facilitated by nickel-agarose chromatography. The WT enzyme (V max ϭ 136 units/mg; K m for HMG-CoA ϭ 26 M) was comparable with the pTrc-HL-1-expressed enzyme. The protein concentration was determined by the method of Bradford (11). The enzyme activity was determined by a citrate synthase-coupled assay (1), modified as described previously (12).
Crystallization, Data Collection, and Structure Determination-WT crystals were obtained by the vapor diffusion method in the presence of 1 mM inhibitor, 3-hydroxyglutaryl-CoA (HG-CoA), as described previously (8). The R41M mutant crystals were obtained under similar conditions, except that the substrate (HMG-CoA) was used in place of the inhibitor (HG-CoA). Small needles formed immediately after the enzyme (6 mg/ml in 20 mM potassium phosphate buffer, pH 7.8) was mixed 1:1 with equilibration buffer consisting of 0.1 M Hepes, pH 7.5, 60 mM MgCl 2 , 5% glycerol, and 15-18% PEG 8000. These needles required 3-4 weeks to grow into large enough crystals for data collection.
Crystals of the WT lyase in complex with the inhibitor HG-CoA were generated by soaking WT crystals in mother liquor supplemented with additional 5 mM HG-CoA for 4 h. Generation of the complexed crystals of the R41M mutant lyase with the substrate HMG-CoA followed the identical procedure, i.e. the mutant crystals were soaked in mother liquor containing 5 mM HMG-CoA. All crystals were soaked in cryoprotectant consisting of mother liquor with an additional 20% glycerol prior to flash-freezing in liquid nitrogen. X-ray data for the WT lyase crystal soaked with the inhibitor HG-CoA (hereafter referred to as WT/HG-CoA) and for the R41M mutant were collected at Ϫ180°C to resolutions of 2.4 and 2.2 Å, respectively, using an in-house R-AXIS IV ϩϩ detector coupled to a Rigaku Micromax 007 x-ray generator operating at 40 kV and 20 mA. For R41M crystals soaked with the substrate HMG-CoA (hereafter referred to as R41M/HMG-CoA), 2.25-Å resolution data were collected using synchrotron radiation at BioCARS beamline 14-BM-C at the Advanced Photon Source, Argonne National Laboratory. Data processing was carried out using the HKL2000 program package. All crystals belong to the monoclinic space group C2, with six monomers in the asymmetric unit and with Matthews coefficients of V m ϭ 2.2 Å 3 /Da, corresponding to 42% solvent content.
The crystal structure of WT/HG-CoA was determined and refined using the WT human lyase structure (Protein Data Bank code 2CW6) (8) as the starting model. Bound HG-CoA was located using the difference Fourier map calculated with the ͉F o ͉ Ϫ ͉F c ͉ coefficient. The R41M mutant crystal structure was determined by molecular replacement with MOLREP within the CCP4 program suite (13) using the monomer structure of WT lyase (Protein Data Bank code 2CW6) as the search model. There is an ϳ2-Å difference in the a axis between the WT/HG-CoA lyase and R41M mutant enzyme crystals. The structure of R41M/HMG-CoA was also solved using the R41M structure by the difference Fourier method. Refinements were carried out using the program CNS (14) and together with manual model adjustments using the program TURBO-FRODO (15) and COOT (16). Water molecules were added at electron densities Ͼ3 in the ͉F o ͉ Ϫ ͉F c ͉ map after several cycles of model building and structure refinement. The final models gave the following crystallographic R-factor/R free values: WT/HG-CoA, 0.211/0.284; R41M, 0.219/0.263; and R41M/HMG-CoA, 0.222/ 0.274. The data collection and refinement statistics are given in Table 1.

RESULTS
Overall Structure and the Ligand-binding Site-We have determined structures of human HMGCL in three different crystal forms: the WT enzyme crystallized in the presence of a competitive inhibitor, HG-CoA (17), and soaked with HG-CoA (WT/HG-CoA); the R41M mutant crystallized in the presence of the substrate HMG-CoA but with no ligand bound (R41M); and R41M soaked with HMG-CoA (R41M/HMG-CoA). In each crystal form, there are six monomers (three dimers) in the asymmetric unit, and not all of the six protomers contain bound ligand. In fact, in the WT/HG-CoA crystal, only one monomer contains HG-CoA, and another monomer contains 3-hydroxyglutaric acid (HGA; presumably the hydrolysis product of HG-CoA). The R41M crystal has no Mg 2ϩ ion bound in any of the six monomers. In the R41M/HMG-CoA crystal, only one monomer contains bound HMG-CoA and Mg 2ϩ , one contains Mg 2ϩ only, and the other four monomers contain no Mg 2ϩ and no ligand. Thus, we have a total of 18 different monomer structures, including the WT with Mg 2ϩ bound (WT-Mg 2ϩ ), R41M with apo structure (i.e. no Mg 2ϩ ion) (R41M), R41M binary complex with Mg 2ϩ ion (R41M-Mg 2ϩ ), and three different ternary complexes (the WT with HG-CoA and Mg 2ϩ , i.e. WT-Mg 2ϩ -HG-CoA; the WT with HGA and Mg 2ϩ , i.e. WT-Mg 2ϩ -HGA; and R41M with Mg 2ϩ and HMG-CoA, i.e. R41M-Mg 2ϩ -HMG-CoA). Because the structures of R41M and SCHEME 1. HMG-CoA lyase reaction scheme.
R41M-Mg 2ϩ are the same, R41M will be used for both structures. Also, as all ternary complexes include Mg 2ϩ , they will be referred to as simply WT-HG-CoA, WT-HGA, and R41M-HMG-CoA. With the exception of some residues (positions 265-271) in the glycine-rich loop that are disordered in some monomer structures, the entire polypeptide starting from the N terminus of the mature protein (Thr 28 ) to Cys 323 (sequence alignment in Fig. 1) is observed in all 18 monomers in the three crystal forms. The last two C-terminal residues, Lys 324 and Leu 325 , are not observed in any of the 18 structures. Because there are no significant differences among the structures of apo and binary complexes of both the WT and R41M with Mg 2ϩ ion (root mean square deviations between pairs of Ͻ0.6 Å for the entire visible 296 C␣ atoms), they will be referred to as WT and R41M. Furthermore, apart from the two regions Ser 131 -Gln 148 and Gly 264 -Gly 274 , the latter of which is disordered in some monomer structures, the overall polypeptide folds are the same among all 18 monomers with root mean square differences ranging from 0.41 to 0.72 Å for 274 C␣ atoms ( Fig. 2A). The structural and functional significance of the movements/disorderedness of these two regions will be discussed below. It is interesting to note that not all 18 monomers contain bound Mg 2ϩ , even though the crystallization media for all lyase crystals contained 60 mM MgCl 2 . Although all of the WT enzyme monomers contain Mg 2ϩ , only two of the 12 monomers in R41M mutant crystals contain the metal ion, including the one that has bound HMG-CoA. Whether the mutant has a lower affinity for Mg 2ϩ is not known. However, the K d value of the mutant for Mn 2ϩ (which binds more tightly than Mg 2ϩ ) is almost the same as that of the WT (2.1 versus 1.8 M) (10). Each monomer of human lyase is composed of nine ␤-strands and 12 ␣-helices and forms a (␤␣) 8 -triosephosphate isomerase (TIM) barrel with an additional helical region, residues 290 -325, as reported previously (8). A structure of the physiological dimer designated on the basis of the extensive intersubunit contact area (8) is shown in Fig. 2B; one subunit is rotated by ϳ90°with respect to the other.
Structures of Ternary Complexes-Electron densities corresponding to the bound ligands are displayed in Fig. 3. Overall, the electron densities are well defined with the exception of that for the adenine ring in the ternary structure of R41M with HMG-CoA (Fig. 3E).
To trap the ternary complex of the WT, the competitive inhibitor HG-CoA was used for the WT lyase. Because our previous co-crystallization attempts using HG-CoA resulted in the lyase complex with a hydrolysis product (HGA) of the inhibitor (8), both co-crystallization and soaking methods were employed. Preformed WT lyase crystals that were obtained in the presence of HG-CoA were soaked further with a solution containing HG-CoA. However, of six monomers (three dimers) in the asymmetric unit, only one monomer of the WT enzyme has a bound inhibitor (Fig. 3, A and D), and the other monomer of the same dimer has bound HGA (Fig. 3C), as observed previously with cocrystallization with HG-CoA (8). The remaining four monomers contain only Mg 2ϩ ion, but no other ligand. In the structure of the monomer that contains HGA and Mg 2ϩ , one of the carboxylates of the hydrolysis product (HGA) is coordinating Mg 2ϩ , in contrast to the structure of the HGA complex obtained by co-crystallization only. In the latter case, the HGA molecule is Ͼ6 Å away from the Mg 2ϩ ion (8). Thus, the HGA binding mode in the active-site cavity is not unique and is quite different from that of the glutaryl moiety of HG-CoA or HMG-CoA (see below). Therefore, model building for the ternary complex structure of a CoA derivative based on the structure of either of these two HGA complexes would not result in a correct binding mode for the substrate or inhibitor. These observations serve as a reminder that one must proceed with caution when modeling a ligand-bound structure on the basis of a reference structure that is missing a significant portion of the ligand. The inhibitor HG-CoA is bound with the acyl moiety adjacent to the Mg 2ϩ activator cation, which is situated near the base of the C-terminal cavity of the barrel ( Fig. 2A  and 3A), as was predicted previously from the WT structure (8). Although crystal growth required the presence of HMG-CoA substrate or HG-CoA analog, soaking the crystals with these compounds was required to improve occupancy and detectable electron density. Nonetheless, the orientation of bound ligand satisfies base-line functional expectations because, as indicated below, the acyl group C3 and C5 oxygens are coordinated with the activator cation. Moreover, only the physiologically active Sisomer of the substrate/analog is bound despite the fact that chemically synthesized mixtures of isomers of HMG-CoA substrate or HG-CoA analog were used for cocrystallization and soaking. The bound ligand adopts an L-shaped conformation; the acylpantetheine moiety forms the long arm, and the 3Ј-AMP forms the short arm, with the pyrophosphate making the right angle turn, resulting in exposure of the 3Ј-AMP to solvent at the surface of the molecule. This conformation corresponds to that observed in the structures of other acyl-CoA-binding enzymes, including the acyl-CoA dehydrogenase family (18) and malate synthase (19,20), and contrasts with the compact bent conformation observed for citrate synthase (21) or the completely extended linear shape observed for methylmalonyl-CoA mutase (22).  4 shows detailed interactions between the bound ligand and the surrounding residues. In the structure of WT-HG-CoA (Fig.  4A), the oxygen atoms of the C5 carboxyl and C3 hydroxyl of the hydroxyglutaryl moiety of the inhibitor are coordinating the activator Mg 2ϩ ion. In addition, there are hydrogen bond interactions from a C5 carboxyl oxygen to hydroxyl oxygens of Ser 169 (2.5 Å) and Thr 205 (2.7 Å). The Asp 42 carboxyl displays an interaction with the C3 oxygen (3.5 Å). The guanidinium nitrogens of Arg 41 closely interact with the C3 (3.0 Å) and C1 (3.3 Å) oxygens, and the Gln 45 amide nitrogen interacts with the C1 oxygen (3.1 Å). Also, a well ordered water molecule is in close proximity to both the C1 (3.3 Å) and C3 (3.8 Å) oxygens and one of the Asp 42 carboxyl oxygens (3.3 Å). In addition to these multiple binding contacts for the acyl group, the CoA moiety makes additional interactions with the polypeptide: the amide nitrogen and the main chain carbonyl oxygen of Asn 138 form hydrogen bonds with the oxygen of the amide that links cysteamine and 4-phosphopantetheine moieties; the Trp 81 indole nitrogen makes a hydrogen bond with a pyrophosphoryl oxygen; the indole ring also flips to shield the pantetheine portion of the CoA moiety from solvent; Lys 111 forms a hydrogen bond with the ribityl 2Ј-hydroxyl oxygen; and Asn 109 makes hydrogen bonds with both the 4-phosphopantetheine-bridging hydroxyl oxygen and N7 of the adenine ring. These latter interactions explain the L-shaped bend observed for binding of the CoA moiety. Furthermore, the interactions involving Asn 138 are at least partially responsible for the bending and pulling of the helix-loop-helix region containing Ser 131 -Gln 148 (Fig. 2A).
Note that interactions between the polypeptide and the pyrophosphoryl adenosine moiety of CoA observed in the structure of R41M-HMG-CoA are slightly different from those seen in the WT-HG-CoA structure, presumably due to the disordered adenine ring of HMG-CoA.
Structure of R41M in Complex with HMG-CoA-To determine the binding mode of the substrate HMG-CoA, the catalytically deficient R41M mutant was employed. The overall binding mode is essentially the same as that of HG-CoA to the WT enzyme (Fig. 3B); however, there are a few subtle but significant differences. Fig. 4B shows the detailed interactions between the substrate and the surrounding residues. In addition to the water coordinating to the Mg 2ϩ ion (W1), a second ordered water (W2 in Fig. 4B) is situated in close proximity (3.2 Å) to the substrate C3 oxygen and is stabilized by interaction with the Glu 72 carboxyl oxygen (2.5 Å). This water (W2) is positioned where the guanidinium group of Arg 41 lies in the WT structure. Thus, two ordered water molecules interact with the substrate C3 oxygen; one (Mg 2ϩ -coordinated) is positioned by Asp 42 (2.8 Å), and the other is situated in the space vacated due to the R41M mutation and stabilized by Glu 72 . A major difference in the HMG-CoA complex is the position of the glycinerich loop, which moves closer to the acyl group of bound substrate by ϳ6 Å. This brings Cys 266 , a residue implicated as functionally important by kinetic, mutagenesis, and affinity labeling studies, into the active site. The thiol group of Cys 266 is now situated 3.4 Å from the C1 carbonyl oxygen of HMG and 4.5 Å from the water molecule that coordinates Mg 2ϩ (Figs. 3B and 4B).
Divalent Activator Mg 2ϩ Ion-binding Site-When the hydrolysis product HGA is bound to the enzyme, i.e. when the bound ligand lacks the CoA moiety, the ligand binds in two different modes. When the HGA concentration is relatively low, the Mg 2ϩ coordination involves His 233 , His 235 , Asp 42 , Asn 275 , and two water molecules, and HGA is not within the coordination sphere. Thus, the HGA ligand is away from the bottom of the active-site pocket (8). On the other hand, when the HGA concentration is higher (this study, due to both cocrystallization and soaking with HG-CoA), the Mg 2ϩ ion coordination is tetrahedral, involving His 233 , His 235 , Asp 42 , and one carboxyl oxygen of HGA. Thus, HGA is positioned deeper into the long narrow cavity of the active site, but not deep enough for both the carboxyl and hydroxyl oxygens of HGA to coordinate to the Mg 2ϩ ion. For the enzyme complex with either HG-CoA or HMG-CoA (i.e. when the CoA moiety is present), the Mg 2ϩ ion has nearly perfect octahedral coordination, involving His 233 , His 235 , Asp 42 , C3, and C5 oxygens from the S-3-hydroxyglutaryl moiety (Fig. 4A) or S-3-hydroxy-3-methylglutaryl moiety (Fig. 4B) and a well ordered water molecule. The S-isomer of HG and HMG moieties is required for Mg 2ϩ coordination of the C3 oxygen. This isomer is selectively bound in these complexes, in agreement with the observation (1) of the stereospecificity of this enzyme. All four amino acids involved in activator cation coordination are invariant residues that have been shown to make substantial contributions to HMGCL function. Interestingly, two other members of the HMGCL TIM barrel family of enzymes, 4-hydroxy-2-ketovalerate aldolase (23) and isopropylmalate synthase (24), also utilize for cat-  AUGUST 20, 2010 • VOLUME 285 • NUMBER 34 ion coordination two histidines and one aspartate; these residues are conserved in the HMGCL family.

Human HMGCL-Mg 2؉ -Substrate/Inhibitor Structures
Role of Lys 48 -Lys 48 of HMGCL has been implicated in an inherited K48N mutation (25). In addition, proteomics results have shown that this residue is a site of post-translational acetylation (26). For these reasons, K48N and K48Q mutant proteins (which model the proposed human mutation and mimic Lys 48 acetylation, respectively) were engineered, isolated, and kinetically characterized ( Table 2) (Figs. 2B and 3B). The distances from both the C␣ and ⑀-NH 2 of Lys 48 to the carbonyl oxygen of the thioester are Ͼ15 Å, and their closest distances to the bound HMG-CoA are also Ͼ14 Å to the amide nitrogen of the 4-phosphopantothenic acid moiety (Fig. 2B). The C terminus of the lyase molecule lies Ͼ25 Å from the adenine ring of the substrate, contrary to the results obtained from the modeled structure of the enzyme-ligand complex (25). Instead, the C terminus is interacting with the glycine-rich loop of the other monomer (Fig. 2B).

Human HMGCL Ternary Complex Structures: Similarities and
Contrasts-Because HMGCL is a single substrate enzyme, to prepare enzyme-Mg 2ϩ -acyl-CoA complexes for crystallization, it was necessary to employ the competitive inhibitor HG-CoA to avoid turnover in the complex with the WT enzyme. Conversely, for a complex with the substrate HMG-CoA to persist, the catalytically deficient R41M mutant protein was used. As discussed in more detail below, the positioning observed for the flexible loop (Leu 263 -Asn 275 ) that harbors the signature sequence for HMGCL is substantially different in these two acyl-CoA complexes. However, regardless of which protein or acyl-CoA molecule is used to detect bound acyl-CoA in these two ternary complexes, observed cation liganding is similar, but it differs from observations made for the enzyme-Mg 2ϩ -3-hydroxyglutarate ternary complex (8). In complexes with either an acyl-CoA substrate or analog, oxygen atoms from both the C5 carboxyl and C3 hydroxyl groups ligand to cation. In the absence of a CoA ligand, Mg 2ϩ is coordinated to a second water molecule and the Asn 275 amide nitrogen, both of which replace the ligand oxygens. In all three cases, the four remaining cation ligands are contributed by Asp 42 , His 233 , His 235 , and an ordered water molecule.
The physiological significance of the observed acyl-CoA binding in both ternary complexes is underscored by the fact that, despite incubation of enzymes with chemically synthesized RS-mixtures of HMG-CoA or 3-hydroxyglutaryl-CoA, the only bound species observed in each complex represents the S-isomer. Stegink and Coon (1) experimentally demonstrated that HMGCL is specific for the S-isomer. Cation coordination to substrate/analog contributes to specificity because coordination to both the C3 hydroxyl and C5 carboxyl oxygens of the acyl moiety could not occur for R-acyl-CoA isomers.
Contrasts are also apparent upon comparison of the acyl-CoA-binding site experimentally determined for both of these ternary complexes with the model predicted on the basis of molecular docking (25). Those computational studies relied on structural coordinates determined for the enzyme-cation-HGA complex (8). Although the structural results indicated the approximate location of the catalytic site, some ambiguity persisted due to the presence of two carboxyl functionalities in bound HGA. Based on the available information, the modeling study imposed the constraint that the HMG-CoA acyl chain dock to the protein surface where HG binding had been observed. Without availability of other information on which carboxyl group of bound HG is originally thioesterified to CoA and without details implicating specific residues in CoA binding, the molecular docking model that resulted suggested that CoA binds in an extended fashion, with the adenosine moiety oriented toward the C-terminal ␣-helical region of the protein.
Interaction of the HMG-CoA 3Ј-phosphoryl with Asn 311 and Lys 313 was proposed. Frameshift mutations in DNA encoding the human enzyme result in C-terminal deletions. Observation of diminished activity for such mutants was interpreted in the context of this docking model of the protein. Additionally, a missense mutation that produces K48N protein was proposed to correlate with inherited metabolic disease. This residue was modeled to suggest interaction of Lys 48 with a pantetheine oxygen of HMG-CoA. Disruption of substrate binding or conformation of bound substrate upon mutation of Lys 48 was therefore proposed (25) as the basis for the observation of diminished mutant enzyme activity. Because the ⑀-amino of Lys 48 is observed to be located Ͼ15 Å from the CoA pantetheine oxygen (Fig. 2B), and the C terminus of the protein is Ͼ25 Å from the adenine moiety of the substrate, other explanations (altered expression, folding, stability, etc.) offered to account for the low activity reported for these inherited HMGCL mutants need to be carefully considered. For example, truncation of the C-terminal sequence could disrupt dimerization and activity. Native active enzyme from eukaryotes and prokaryotes (9,12,27,28) has been characterized as a dimer of identical subunits.
Interpretation of Functional Observations in the Context of Ternary Complex Structure-HMGCL functions in eukaryotes and prokaryotes, catalyzing key reactions in ketone body biosynthesis and leucine catabolism. Reaction products support metabolic energy production (acetoacetate) and anaplerotic metabolism (acetyl-CoA). The initial identification of functional residues at the active site of the enzyme (29) was accomplished by affinity labeling of the Pseudomonas mevalonii enzyme using 2-butynoyl-CoA to covalently modify the protein. This reagent exhibits K i values of 65 and 320 M for the bacterial and animal enzymes, respectively. The cysteine (Cys 237 ) that was identified in the modified P. mevalonii enzyme maps as Cys 266 in the human enzyme; this residue is conserved in all HMGCL proteins. Subsequent mutagenesis of this residue in recombinant forms of human (30) and bacterial (31) enzymes resulted in decreases in catalytic activity of 4 orders of magnitude upon substitution of cysteine with alanine.
Knock-out of the gene in animals (4) causes prenatal lethality. Human disease results from a variety of inherited HMGCL mutations. Consequently, our functional investigations have focused not only on conserved residues identified by protein modification approaches but also on those point mutations that map to invariant residues in HMGCL proteins. We have modeled such mutations in a recombinant form of the human enzyme and characterized the purified mutants in an attempt to test enzymatic function of the mutated residues. A combination of chemical modification results and characterization of proteins mutated at these sites indicated functional roles for invariant residues His 233 and His 235 , which were subsequently identified in initial structural work (8,32) as activator cation ligands. Other human mutations implicated invariant residues  Asp 42 and Arg 41 . Characterization of purified proteins containing mutations of each of these side chains (10, 33) indicated major decreases (4 and 5 orders of magnitude, respectively) in catalytic activity. Moreover, using the acetyldithio-CoA (an analog of the reaction product acetyl-CoA) to measure product enolization, Arg 41 mutation was shown to eliminate this partial reaction (10). The ternary complex structure of the WT enzyme with Mg 2ϩ and the inhibitor HG-CoA (Fig. 3A) is particularly informative in demonstrating the proximity (3.3 Å) of the Arg 41 guanidinium moiety to the thioester carbonyl oxygen of bound acyl-CoA (Fig. 4A). A similar interaction of arginine with bound acyl-CoA has been suggested in work on the related malate synthase reaction (20). Interestingly, residues corresponding to Arg 41 , Asp 42 , His 233 , and His 235 of the human enzyme are conserved in other members of a broader HMGCL family of proteins such as isopropylmalate synthase, homocitrate synthase, and hydroxyketovalerate aldolase. All of these enzymes utilize acetyl-CoA as a substrate or product. In contrast, only HMGCL proteins contain the affinity label-targeted cysteine in a flexible loop (Leu 263 -Asn 275 ) that harbors a highly conserved "signature" sequence for this protein. Upon substrate binding, this loop moves over to the C-terminal end of the TIM barrel of protein that contains the active site, including the invariant residues identified above. Another factor to be considered in correlating the tertiary complex structural information with functional observations involves reaction stereochemistry. Cleavage of substrate by HMGCL occurs with incorporation of deuterium from solvent D 2 O into the methyl group of acetyl-CoA and inversion of stereochemistry at the substrate C3 (34). In this respect, the stereochemistry of this reaction is in accord with other enzymecatalyzed Claisen condensations or cleavages (35). This observation requires that deprotonation of the substrate C3 alcohol and protonation of the carbanion produced upon C2-C3 bond cleavage occur from the same side of the substrate; this is the side involved in coordination of C3 and C5 oxygens to the activator cation (Fig. 3). An ordered water molecule, as well as two amino acids (Asp 42 and Cys 266 ) with side chains shown to have a large influence on reaction rate, would qualify for participation in these steps based on stereochemical considerations and on the orientation of these candidates in the structures of the HMG-CoA-and HG-CoA-containing ternary complexes (Fig. 3).
Candidates for Functional Roles in Substrate Cleavage-The reaction is critically dependent on the presence of a charge sink for the electron density generated as a result of substrate C2-C3 bond cleavage. Clearly, the interaction of the guanidinium group of Arg 41 with the C1 carbonyl oxygen satisfies this requirement. There are several candidates for participation as a general acid or base in HMG-CoA cleavage, so these assignments are more complicated. Candidates include Asp 42 and the ordered water molecule situated in closest proximity to C2 and C3 of bound acyl-CoA in the ternary complex structures (Figs. 3 and 4). In these structures, the carboxyl of Asp 42 (elimination of which decreases activity by Ͼ10 4 -fold) is in close proximity to the bound ligand C3 hydroxyl oxygen (3.5 Å), but so is the ordered water oxygen, which is positioned 3.3 Å (Fig. 4B) or 3.8 Å (Fig.  4A) away from this C3 oxygen in the ternary complex with HMG-CoA or HG-CoA, respectively. This solvent oxygen is closer to the ligand C2 (4.0 Å) than is the Asp 42 carboxylate (5.6 Å). The requisite proton abstraction from the substrate and the carbanion quenching by protonation of the cleavage product by either candidate (as well as by Cys 266 ) would be compatible with observed reaction stereochemistry. The participation of ordered water in shuttling a proton between the substrate and an active-site residue side chain has precedent in related aldolase reactions. More specifically, in 2-keto-3-deoxy-6-phosphogluconate aldolase (36), the shuttle involves an ordered water and the carboxyl group of a glutamate implicated in catalysis.
Consideration of candidates for participation as active-site acid/base residues (Fig. 5) should, however, also take into account the measurement of pK a ϭ 8.0 in the pH/rate profile for the WT enzyme (30). No titratable group is detectable when the comparable experiment is performed using C266S, suggesting that the sulfhydryl of Cys 266 accounts for the observed pK a , and therefore, its participation in reaction chemistry is important. Moreover, a residue corresponding to this cysteine is covalently modified by the affinity label 2-butynoyl-CoA, demonstrating that, in solution, close juxtapositioning of Cys 266 to bound acyl-CoA can occur. Assignment of a role for Cys 266 becomes complicated because of the considerable mobility of the Cys 266 -containing flexible loop that is evident from our structural results (Fig. 2). In the R41M-containing complex with the substrate, Cys 266 is in closer proximity to bound acyl-CoA than in structures of WT enzyme complexes with hydroxyglutarate or HG-CoA. Although loop positioning in the R41M mutant structure may not reveal the closest possible proximity of Cys 266 to the substrate, it reflects the most relevant experimental structural results available. In the R41M structure (Fig. 4B), Cys 266 is somewhat remote from the substrate C3 hydroxyl oxygen (5.8 Å) but closer to the C1 carbonyl oxygen (3.4 Å) and the ordered solvent oxygen (4.5 Å).
Although both Asp 42 and Cys 266 are plausible participants in a water-mediated proton shuttle, the simplest model (Fig. 5) FIGURE 5. Proposed reaction mechanism for HMGCL. This schematic drawing shows functionally important active-site side chains, Mg 2ϩ , an ordered water molecule, and the HMGCL substrate and products. The left side of the diagram indicates the six Mg 2ϩ coordination ligands, as found in the R41M-Mg 2ϩ -HMG-CoA ternary complex. In the absence of the HMG-CoA or HG-CoA ternary complex ligands, the substrate C5 carboxyl and C3 hydroxyl oxygen ligands are replaced with water and Asn 275 oxygens (not shown) (8).
that is in accord with both the mechanistic and functional data as well as the ternary complex structural results involves participation of the aforementioned ordered water molecule in a proton shuttle with Cys 266 functioning as the partner in the relay. Interactions with both Mg 2ϩ and Asp 42 precisely position this solvent molecule both in close proximity to substrate and 4.5 Å from the sulfur atom of Cys 266 in the mobile loop. Thus, water-mediated deprotonation of the substrate C3 hydroxyl can be supported by the cysteine thiolate anion. Subsequently, the Cys 266 thiolate anion re-forms upon shuttling of a proton from the neutral thiol to the hydroxyl anion that develops after an ordered solvent proton is used to quench the carbanionic form of the acetyl-CoA reaction product.