Isocitrate Dehydrogenase
Created by Brian Kwak
Isocitrate dehydrogenase (PDB ID = 1AI2) is an enzyme involved in the citric acid cycle with a molecular weight and isoelectric point of 45756.71 Daltons and 5.15, respectively. The citric acid cycle is a critical series of enzyme-catalyzed chemical reactions in which living cells undergo during aerobic respiration. The citric acid cycle is crucial to aerobic respiration because the high energy electrons from the carbon compound are transferred to nicotinamide adenine dinucleotide (NAD+), creating NADH. During oxidative phosphorylation, these high energy molecules (NADH) are oxidized to drive the synthesis of adenosine triphophate (ATP). Once citrate is isomerized to isocitrate by aconitase in a two-step process, isocitrate undergoes oxidative decarboxylation, a regulated reaction, to yield alpha-ketoglutarate (Reaction Scheme) (1). As a result, NAD+ is reduced to NADH in the isocitrate dehydrogenase reaction (1).
Isocitrate dehydrogenase (IDH) acts as a catalyst in the oxidative decarboxylation of isocitrate during the citric acid cycle. Because NADH is produced from the reaction, IDH is the first connection between the TCA cycle, electron-transport pathway, and oxidative phosphorylation (1). The allosteric inhibitors of the reaction are NADH and ATP while adenosine diphosphate (ADP) acts as an allosteric activator by lowering the Km for isocitrate by a factor of 10 (1). IDH is dependent on NADP as a coenzyme in order to catalyze this reaction (2). Eukaryotic NADP-IDHs are located primarily in the cell cytosol and mitochondria. IDH can also be found in peroxisomes of human, rat, and mouse cells because the cytosolic NADP-IDHs of these species contain a type 1 peroxisomal targeting sequence at the C terminus (2). IDH catalyzes the oxidation and decarboxylation of isocitrate to alpha-ketoglutarate (AKG) in two distinct steps (2,3,4). The two steps of the reaction begin with the oxidation of isocitrate producing the oxalosuccinate intermediate. This intermediate undergoes decarboxylation and forms AKG (3,4). In addition to taking a major role in the citric acid cycle, NADP-IDHs are involved in cellular defense against oxidative damage, detoxification of reactive oxygen species, and synthesis of fat and cholesterol through the production of NADPH and AKG (2).
DALI Server analysis was conducted to determine if any proteins have similar tertiary structures to IDH. The DALI Server results yielded many proteins with root mean squared deviations (rmsd) less than 10 and Z scores greater than 2, indicating that these proteins have similar tertiary structures with IDH.
3-Isopropylmalate dehydrogenase (PDB ID = 1OSJ, IMDH) is an enzyme which catalyzes a reaction in the leucine biosynthetic pathway, yielding 2-ketoisocaproate from 3-isoproplymalate (5). According to the DALI results IMDH yielded a Z score of 39.1 and a rmsd of 2.2, validating that this protein has similar tertiary structure to IDH. Similar to IDH's effect on the citric acid cycle, IMDH is important in producing high energy molecules, NADH, for the leucine biosynthetic pathway (6).
Homoisocitrate dehydrogenase (PDB ID = 1XOL, HICDH) is a second protein which has similar tertiary structure to IDH. According to the DALI results, HICDH yielded a Z score and rmsd of 39.8 and 2.0, respectively. Although the rmsd value is not greater than 2, HICDH is a member of the beta-decarboxylating dehydrogenases which include IDH and IMDH (7). Thus, the structures of these proteins are related and similar. HICDH is an enzyme involved in lysine biosynthesis through alpha-aminoadipate (7). HICDH functions similarly to IDH by catalyzing a reaction in which high energy molecules are produced.
The structure of IDH has been found to be a dimer of identical 416-residue subunits (3,9). Alternate conformations of IDH have not been determined. The protein structure of IDH cannot be overlooked because the function of the enzyme is a direct result of the structure. Although IDH is composed of only one unique subunit, the dimer of identical 416-residue subunits allows for the protein to be a vital enzyme in the citric acid cycle in Escherichia coli (E. coli).
Three domains reside in IDH: a large alpha + beta domain, a small alpha/beta domain, and an alpha/beta clasp-like domain which involves both subunits (9,10). The large and small domains are composed of a 12-stranded beta-sheet (9). The large and small domains are separated by a distinct cleft and the domains are folded in such a way that they form hydrophobic cores (9). As a result of the cleft, two pockets form between the large and small domain on both sides of the sheet which are referred to the "front" and "rear" pockets (9). The small domain is a typical alpha/beta sandwich structure which consists of residues 125-157 and 203-317 while the large domain consists of residues 1-124 and 318-416 (9). The large domain is made up of a helical subdomain and a large alpha/beta subdomain (9). The clasp-like domain is formed by residues 158-202 of both subunits interlocking (9). Consequently, two strands from both subunits form a four-stranded antiparallel beta-sheet (9). The dimer structure allows for a hydrophobic core to be formed due to the packing of the alpha-helices and beta-sheets (9). Overall, the secondary structure of IDH is 46% helical, 22% sheet, 19% coil, and 12% turn (9).
The most important residue in this dimer is serine-113 (Ser-113) because the enzyme is completely activated by phosphorylation of this active site (3,9,10,11,12,13). The active site lies in the pocket between the large and small domains of IDH, specifically at the edge of the front pocket(9,12,13). Isocitrate or Mg2+-isocitrate will bind at this active site and hydrogen bonds will form between isocitrate and serine 113, arginines 119, 129, and 153, tyrosine 160, lysine 230' (' indicates a residue on the second subunit, contact area), and five water molecules (12,13). Of the five water molecules that form hydrogen bonds with isocitrate, three of them (560-562) are unique to the substrate-bound structure while waters 458 and 479 are present in the unliganded structure of IDH (13). The active site Mg2+ is coordinated to six oxygen ligands by isocitrate, aspartic acid 283 and 307, and two bound water molecules in an octahedral arrangement (12,13). The coordination of Mg2+ to the alpha-carboxylate and the hydroxyl oxygen of isocitrate is crucial because this arrangement allows the metal ion to play a significant role in catalysis (12). Nicotinamide adenine dinucleotide phosphate (NADP+) also binds in the same cleft as isocitrate which is between the small and large domains of the protein (13). The adenine functional group binds with the binding site consisting of the side chains of Ile-37, Ile-320, His-339, Ala-342, and Val-351, the aliphatic portion of the side chains N-352 and D-392, and the main chain at G-321 and N-352 (13). These residues make up the contact area. There is a hydrogen bond made between nitrogen (N6) of adenine and the main chain carbonyl oxygen of residue 352 of isocitrate (13).
The significance of the two identical subunits is that it provides for the active site which is critical in catalyzing the oxidation and decarboxylation of isocitrate to produce alpha-ketoglutarate (1). The oxidation of isocitrate by IDH is a two step process which begins with isocitrate binding to the active site of IDH. This allows isocitrate to be oxidized to oxalosuccinate by removing a proton from the hydroxyl oxygen to a base, aspartate-283', and transferring a hydride to NADP+ (13). The location of aspartate 283' relative to the active site is important because it is the closest base to the alpha-hydroxyl oxygen which is favorable for a proton transfer (13). In the second step of the reaction, carbon dioxide is produced as a byproduct as the beta-carbon of the oxalosuccinate is protonated to form alpha-ketoglutarate (13). The structure of IDH is important because tyrosine-160 and lysine-230' form hydrogen bonds with the beta-carboxylate of isocitrate (13). As a result, the hydrogen bonds form a compound which can serve as the acid catalyst that protonates carbon 3 after decarboxylation (13). The importance of Mg2+ in the function of the enzyme is that its position stabilizes the negative charge formed on the hydroxyl oxygen during dehydrogenation (13). Specifically, the metal ion is hypothesized to be coordinated by the keto oxygen and the adjacent alpha-carboxylate which allows it to stabilize the formation of an enolate intermediate (13). Thus, Mg2+ is important in stabilizing negative charge in both steps of the reaction. It should be noted that IDH has not been targeted by a drug.
HICDH is a member of the beta-decarboxylating dehydrogenases which include IDH and 3-isopropylmalate dehydrogenase (IPMDH) (7). Therefore, HICDH shows structural similarities to IDH. Like IDH, HICDH catalyzes a reaction essential for lysine biosynthesis by utilizing isocitrate as a substrate (7). HICDH is composed of two domains that are separated by a pocket which is similar to that of IDH (7). Although the active site (Tyrosine 125) of HICDH differs from IDH, many of the residues that recognize similar functional groups are conserved such as R-88, R-98, R-118, K-171, D-204, D-228, and D-232 (7). All in all, the structural similarities between these proteins can explain the similar functions.