2-Oxoglutarate Dehydrogenase
Created By: Matthew Perez
The enzyme 2-oxoglutarate dehydrogenase E1o (E1o) (PBD ID= 2JGD) is one of three enzymes that make up the 2-oxoglutarate dehydrogenase multi-enzyme complex (OGDH) (Frank et al., 2007, p. 640). The other enzymes are E2 and E3. The OGDH acts as a rate limiting step in the tricarboxylic acid cycle or Krebs cycle (Frank et al., 2007, p. 639). The Krebs cycle, part of the glycolytic pathway, is a series of reactions under aerobic conditions in which pyruvate is converted to acetyl-Co-A and oxidized to CO2. ATP is synthesized by oxidative phosphorylation from the products of this reaction (Garrett & Grisham, 2012 p. 607). E1o is responsible for the oxidative decarboxylation of α-ketoglutarate, an intermediate in the Krebs cycle, ultimately leading to the formation of Succinyl-CoA, an intermediate, after further modification by the E2 and E3 components (Frank et al., 2007, p.639). During this process CO2 is released and NAD+ is reduced to NADH. This reaction occurs at the fourth step of the Krebs cycle (Garrett & Grisham, 2012 p.612). This enzyme is also notable in that it has been recognized that OGDH activity is significantly lowered in some neurodegenerative disorders such as Parkinson’s disease (Gibson et al., 2000, pp.104-105; Tretter & Adam-Vizi, 2005, p.2336). Before the work of Frank et al the structure of E1o was the only component of the Krebs cycle that had not been structurally characterized. Frank et al studied E1o in Escherichia coli. According to the EXPASY server the E1o component is a 106061.72 Da protein with an isoelectric point of 6.04 (Artimo et al, 2012).
The structure of E1o is responsible for its catalytic activity. The enzyme is a globular homodimer consisting of one α subunit and one β subunit; each is 933 residues in length (Frank et al., 2007, p,649). The alpha subunit is 37% α-helices and 12% β- sheets while the β subunit is 38% α-helices and 13% β-sheets (PDB ID: 2JGD). Each subunit also includes beta turns and random coils. These two subunits interface to create an active site where the catalytic reaction occurs.
The primary function of E1o is to catalyze the oxidative decarboxylation of α-ketoglutarate. E1o is dependent on the coenzyme thiamine diphosphate (ThDP), also known as thiamine pyrophosphate, to perform the decarboxylation reaction. ThDP binds to E1o; activating ThDP. ThDP removes a carboxylate group from α-ketoglutarate resulting in an intermediate that reacts with the coenzyme CoASH to form Succinyl-CoA (Garrett & Grisham, 2012 p. 622; Yeaman, 1989, pp. 625-626). The most important function that E1o performs is binding ThDP at the active site. In E1o there is a residue, Glu-580, which is thought to be responsible for activating ThDP (Frank et al., 2007, pp. 642-643). Glu-580 also marks the entrance to a channel between the two subunits. This channel forms the active site. There are three residues from each subunit that appear along the sides of the channel. These residues are Glu-368, Glu-580, and Glu-662. There are also three additional residues, His-260, His-298, and His-729 that are found in close proximity to the cavity (Frank et al., 2007, p. 643). Because of their location these His residues are thought to help define the ThDP binding pocket. Additionally, there are two active site loops that are present in each subunit. These loops are made up of residues 391-407 and 458-471 (Frank et al., 2007, pp. 643-644). The loops are believed to help bind ThDP. Divalent magnesium is also necessary in order for the enzyme to function properly. Mg2+ binds to the active site near ThDP but it is not clear exactly where (Frank et al., 2007, p. 642).
According to Frank et al, the ThDP binding site can be inhibited by the dicarboxylic acid, oxaloacetate. Oxaloacetate is a naturally occurring constituent found in the Krebs cycle. Oxaloacetate can bind to the active site by binding to His-260, His-298, and His-729 found near the cavity. By binding to these sites oxaloacetate obstructs the active site and inhibits the catalytic function of E1o (Frank et al., 2007, p. 645). When physiologic levels of oxaloacetate were added to E1o significant inhibition of the enzyme was observed. This suggests that oxaloacetate plays a regulatory role in the Krebs cycle by means of “cross regulation” since it is naturally present in the cycle ((Frank et al., 2007, p. 645).
In addition to the active site there is a ligand binding site that is found away from the active site (Frank et al., 2007, pp. 645-646). The ligand is adenosine monophosphate (AMP). This binding site is present in both subunits. AMP binds to E1o by forming hydrogen bonds between the phosphate group of AMP and residues Arg-337, Arg-710, and His-313 (Frank et al., 2007, pp.645-646). Frank et al tested E1o to determine the role the AMP binding site plays. They concluded that the presence or absence of AMP had neither an inhibitory nor activating effect regarding E1o’s catalytic abilities. Several different adenosine molecules were tested in place of AMP and the AMP binding site was mutated, but no effect was observed. So, the function of this binding site is still yet to be determined pending further investigation.
Each subunit also has an N-terminal “tower domain” that is believed to be responsible for interacting with the E2 component of OGDH. This domain consists of four alpha helices, two from each subunit, formed by residues 85-190 (Frank et al., 2007, p. 641). This domain is important because it allows the products of reactions to be transferred between different components of the enzyme without undergoing side reactions (Frank et al., 2007, p. 641; Tretter & Adam-Vizi, 2005, p. 2335).
The pyruvate dehydrogenase complex (PDH) is similar to OGDH in its structure and function; both play an important role in the Krebs cycle and both contain E1, E2, and E3 components (Garrett & Grisham, 2012 p. 613). PDH links glycolysis and the Krebs cycle while OGDH performs a rate limiting step in the Krebs cycle (Frank et al., 2007, p. 639; Tretter & Adam-Vizi, 2005, p. 2336). Pyruvate dehydrogenase E1 is similar to 2-oxoglutarate E1 in that both are responsible for catalyzing oxidative decarboxylation reactions by binding to ThDP (Garrett & Grisham, 2012 p. 622; Kato et al., 2008, pp. 1849,1856).
When 2-oxoglutarate E1from Escherichia coli and pyruvate dehydrogenase E1 from Homo sapiens (PDB- 1NI4) were compared using the BLAST server there was a 19% match of the sequence; the E-value was 0.0002. BLAST compares the primary structure of the query protein to other known proteins. An E value of less than 0.05 suggests significant similarity between the sequences of two proteins (Altschul et al, 1990). From the BLAST results it can be seen that these two proteins are very similar. These two proteins were also compared using the Dali server which compares the tertiary structure of proteins. A Z-score greater than 2 means that two proteins have similar folds (Holm, 2010). The Z-score when comparing these two proteins was 60.1. This suggests that these two proteins have similar folds. In this case the folds are characteristic of those found in other thiamine dependent enzymes (Frank et al., 2007, p. 640).
One difference between the two proteins is their subunit structure. As previously stated 2-oxoglutarate dehydrogenase E1 is a homodimer. Pyruvate dehydrogenase E1 is a α2β2 heterotetramer. This means that the molecule contains two subunits. Each subunit contains one α and one β chain (Ciszak et al., 2003, p. 21240). The alpha subunits each contain one six stranded parallel β-sheet and 10 α-helices in addition to a 28 residue N-terminal fragment and a 36 residue C-terminal fragment. The β subunits contain two domains that consist of one six stranded parallel β-sheet with seven α-helices in one domain and one four stranded parallel β-sheet with four α-helices and one antiparallel β-strand in the other domain (Ciszak et al., 2003, p. 21242). The α subunit is 41% α-helices and 14% β-sheets while the β subunit is 39% α-helices and 16% β-sheets (PDB ID: 1NI4). This distribution is similar to the distribution found in the two subunits of 2-oxoglutarate E1o. So, although their subunit structure differs, the secondary structure is very similar.
The two proteins also differ in that pyruvate dehydrogenase E1 has two active sites whereas 2-oxoglutarate dehydrogenase only contains one active site (Ciszak et al., 2003, pp. 21240-21241; Frank et al., 2007, p. 641). This is likely due to the differing subunit structure. Since pyruvate dehydrogenase E1 is a heterotetramer it is possible that more binding pockets form as a result of more interfaces between the subunits.
While these two proteins do differ in the number of subunits and the number of active sites they contain, they are similar in their primary structure, secondary structure and their folding patterns. As a result, they both carry out similar catalytic functions by binding to ThDP.