Human_Pyruvate_Dehydrogenase_E1

Human pyruvate dehydrogenase E1 component (PDB ID: 6CFO) from Homo sapiens is part of the pyruvate dehydrogenase multienzyme complex (PDHc). It connects glycolysis to the tricarboxylic acid cycle by producing Acetyl-CoA via the decarboxylation of pyruvate, thus playing an important role in glucose metabolism. This protein is of significant interest to scientists, because the modulation of reversible phosphorylation of this protein can be used to treat cancer, diabetes, and obesity (1,2). Pyruvate is the product of glycolysis, and it must experience modifications to maximize the yield of ATP for energy. Pyruvate initially undergoes a decarboxylation reaction to form Acetyl-CoA which is catalyzed by the PDHc.

Acetyl-CoA is synthesized in the mitochondrial matrix indicating that the pyruvate dehydrogenase complex must be located inside of the mitochondria. This implies that the primary structure of the protein contains a signal sequence that leads it to its destination. The final primary structure after the proteins translocation also does not contain methionine on the N-terminus suggesting that the signal sequence must have been cleaved by a protease once it has reached the mitochondria (2). The aqueous environment in which the protein functions reveals that the protein complex has a simple primary structure that has polar residues facing the aqueous solution while having non-polar residues within its core.

The secondary structure of this protein contains α-helices, β-sheets, and random coils. The α-helices comprise 41% of the structure for the α-subunits and 38% for the β-subunits. The α-helices on the surface are amphipathic with alternating polar and non-polar residues while the helices that are tucked within the protein are completely hydrophobic with only non-polar residues (2). β-sheets comprise 15% of the structure for the α-subunits and 16-17% of the β-subunits. The β-sheets reveal the same pattern shown with the α-helices in that there are amphipathic and hydrophobic β-sheets. Random coils make up 44% of the α-subunits and 54-55% of the β-subunits (2).

The tertiary structure of pyruvate dehydrogenase must be viewed in subunits. The α-subunits contain 365 residues, and the β-subunits contain 341 residues (2). Individually, these subunits form globular proteins. The most important aspect of the protein is revealed in its quaternary structure. Pyruvate dehydrogenase is a heterotetramer of two α-subunits and two β-subunits which forms a globular protein with a total of four subunits. The α and β-subunits are almost identical with only slight variations. The α-subunits are the catalytic subunits while the β-subunits are scaffolding subunits. The molecular weight of the pyruvate dehydrogenase E1 complex is 155.87 kD, and it has an isoelectric point of 6.24 (4). The full protein structure is known through X-ray diffraction. Ligands were also not employed to induce crystallization (2,3).

The pyruvate dehydrogenase E1 complex functions by catalyzing the reaction between thiamine pyrophosphate (ThDP) and pyruvate. This enzyme is not regulated by allosteric interactions nor any inhibitory proteins indicating that PDHc is constitutively active. A cofactor, Mg2+, interacts with ThDP to mediate the decarboxylation reaction. The structure of pyruvate dehydrogenase E1 component reveals a crevice that exposes the α-subunit active site to the matrix of the mitochondria to allow for substrate binding (1). Mg2+ is located within the active site and forms a 6-bond coordination complex that uses ThDP to facilitate this oxidative reaction. It is stabilized by αAsn-196, αTyr-198, and αAsp-167 through polar or electrostatic interactions. Additionally, Mg2+ anchors ThDP in the correct position to interact with pyruvate (1). The active site also contains αGlu-580 which activates ThDP making it more reactive with pyruvate. In addition to αGlu-580, the active site contains αHis-260, αHis-298, αHis-729, αGln-685 and αSer-321 which provide specificity for the substrates (1). The random coils of the α-subunits that are near the active site play a critical role that scientists believe will allow humanity to have a better grasp of glucose metabolism.

The random coil from the α-subunits located near the active site is called phosphorylation loop A. This loop contains αArg-259 and αHis-263, which serve as key anchors for ThDP by forming indirect hydrogen bonds with the phosphate group (3). Phosphorylation loop A's αSer-264 has a hydroxyl group directly pointing towards the active site (5). This residue can be phosphorylated, causing a conformational change that abolishes the binding of the E2 complex of pyruvate dehydrogenase resulting in the protein's inactivation (5). The phosphorylated serine stops the catalysis at the pre-decarboxylation intermediate stage by preventing the E2 complex from interacting with the substrate on the E1 component. This modification also increases the steric hindrance between the loop and active site which decreases the binding affinity for ThDP. This impediment prevents αArg-259 and αHis-263 from interacting with the substrate which decreases the number of bonds that hold it in place (3). Phosphorylation loop A is initially ordered in the structure, however, the αpSer-264 causes the loop to become disordered which may play a role in the binding affinity of ThDP (3). Scientists are unsure whether this ordered to disordered conformation plays a role in the inhibition of PDHc's enzymatic activity (5). Better understanding of this process can lead to more control of glucose metabolism which can lead to treatments for cancer, obesity, and diabetes (1).

Cancer cells require a large amount of ATP in order to supply their need to undergo constant mitosis. Their demand for energy is significantly higher than traditional somatic and neuronal cells. By inactivating PDHc in cancer cells, physicians and scientists can selectively kill them. In obesity and diabetes, proper sugar intake and utilization are the underlying issues with these diseases. While PDHc's function may not directly attribute to these diseases, study of this enzyme helps researchers to better understand glucose metabolism which may lead to treatment (1).

Human pyruvate dehydrogenase E1 component has many similarities to 2-oxoglutarate dehydrogenase from Escherichia coli (PDB ID: 2JGD). PSI-BLAST is a program that statistically analyzes the degree of similarity between two proteins' primary structure by assigning an E value. A value less than 0.05 indicates significant similarity, and the E value for 2-oxoglutarate dehydrogenase was 1e-52 (6). Since prokaryotes do not have any specialized organelles, 2-oxoglutarate dehydrogenase is found in the cytoplasm of the bacteria. The major difference between the two proteins is that the α and β-subunits are covalently fused into a single polypeptide rather than weakly associated as in human PDHc (7). This single polypeptide chain contains 933 residues, and contains significantly more hydrophilic regions and residues compared to its human analog. 2-oxoglutarate dehydrogenase also contains a total of 1866 residues compared to pyruvate dehydrogenase's 1412 (8).

The secondary structure is quite similar with 37% alpha helices, 12% beta sheets, and 51% random coils (8). The slight increase in random coils and hydrophilic regions may indicate the reason for the crystal structure of the protein to be incomplete (8). 2-oxoglutarate dehydrogenase is more disordered than pyruvate dehydrogenase.

The tertiary structure of the two proteins can be compared by using the Dali server. The Dali server compares two tertiary structures of proteins which result in a Z-score. Z-scores are calculated from the similarities in protein intramolecular distances using the sum-of-pairs method. A Z-score that is above 2.0 represents structural similarity, and the score for 2-oxoglutarate dehydrogenase was 20.6 (9). Since 2-oxoglutarate dehydrogenase is composed of two similar subunits, it is called a homodimer. While the primary structure and tertiary structure are similar, the crucial similarity lies in their function. Similar to PDHc, 2-oxoglutarate dehydrogenase catalyzes the rate limiting step of 2-oxoglutarate to succinyl-CoA in the Krebs cycle (7).

PDHc is dependent on ThDP to oxidize pyruvate to Acetyl-CoA. Similarly, 2-oxoglutarate dehydrogenase utilizes ThDP to oxidize 2-oxoglutarate to succinyl-CoA. Substrate specificity of the enzyme is mediated by three histidine residues exactly as substrates bound to pyruvate dehydrogenase (7). Although the tertiary structure may be similar, the major differences come from the distances of the domains that are bound to the complexes. 2-oxoglutarate dehydrogenase has three components: E1, E2, and E3. While this is similar to its human analog, there is a larger annular gap between the core E2 and peripheral E1 and E3 components (7). The N-terminus of 2-oxoglutarate dehydrogenase actually extends further than pyruvate dehydrogenase's N-terminus. Nonetheless, 2-oxoglutarate dehydrogenase's contrasting quaternary structure still achieves the same rate-limiting, ThDP-dependent oxidative reaction as PDHc's. The oxidative reaction is similar, because a CoA molecule becomes covalently linked to 2-oxoglutarate and pyruvate for 2-oxoglutarate dehydrogenase and PDHc respectively (7).

Pyruvate dehydrogenase E1 component is the catalytic domain for the oxidation of pyruvate to Acetyl-CoA. This oxidation is biologically vital for continuing aerobic respiration which provides energy to cells. Without this protein, cells will lack the ability to maximize their yield of ATP. Absence of PDHc's function also causes aerobic cells to experience anaerobic respiration which produces lactic acid, subsequently resulting in lactic acidosis (1). The random coils located near the active site on the α-subunits (phosphorylation loops A) provide scientists the ability to modulate PDHc's function through reversible phosphorylation. By phosphorylating αSer-264, scientists can inhibit the activity of PDHc.