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Home , Pyruvate dehydrogenase

جامعة تكريت كلية العلوم قسم علوم الحياة الدراسات العليا/ الماجستير

PYRUVATE

اعداد الطالبة عائشة صالح عزيز فرع الحيوان/اختصاص الفسلجة

بإشـــراف الدكتور عمار

Function of pyruvate dehydrogenase The pyruvate dehydrogenase complex (PDH or PDC) is a key regulatory site in cellular , by linking the and subsequent oxidative with and , as well as with both and amino acid metabolism. When stores are reduced in mammals, PDH activity is regulated downward to limit the use of by oxidative phosphorylation in tissues that can use fatty acids or ketone bodies, such as heart and skeletal muscle. The important exception is neuronal tissue, which processes glucose almost exclusively for ATP production. Activation of PDH both facilitates use of carbohydrate to meet energy demands and also converts surplus dietary to fatty acids for longer term energy storage. Perturbation of the regulation of this choice of glucose or fatty acids as energy source is a key part of diabetes, metabolic syndrome and obesity, while metabolic substrate switching from oxidative phosphorylation to glycolysis defines the cancer phenotype, hence recent renewed interest in PDH activity and regulation .

Structure and Activity The pyruvate dehydrogenase complex is a 9.5 megadalton assembly of four : pyruvate dehydrogenase (E1), dihydrolipoamide acyltransferase (E2), dihydrolipoyl dehydrogenase (E3), and one structural (E2/E3 binding protein). The E1 is a heterotetramer of two α and two β subunits. PDH component proteins are arranged as a core of 60 E2 subunits around which are distributed 30 copies of E1, 12 copies of E3, and 12 copies of the E2/E3 binding protein. PDH catalyzes irreversible oxidative of pyruvate to acetyl , as shown in Figure 1.

Figure 1. Five sequential reactions of PDH-catalyzed oxidative decarboxylation of pyruvate to Acetyl Coenzyme A.

Crystallographic structure of pyruvate dehydrogenase Crystallographic structure of pyruvate dehydrogenase (PDH). PH is a six domain dimer with α (blue), α’ (yellow), β (red), and β’ (teal) regions denoted by the different colors. pyrophosphate (TPP) is shown in grey ball and stick form, two magnesium ions in purple undergoing metal ligation with the TPP, and two potassium ions in orange

Figure 2. Crystallographic structure of pyruvate dehydrogenase

Regulation of PDH Activity by Phosphorylation

Not surprisingly given its central role in metabolism, PDH is under tight and complex regulation, which includes regulation by reversible phosphorylation in response to the availability of glucose. In humans, PDH activity is inhibited by site-specific phosphorylation at three sites on the E1α subunit (Ser232, Ser293 and Ser300), which is catalyzed by four different pyruvate dehydrogenase (PDK1-4). Each of the four kinases has a different reactivity for these three sites. Interestingly, phosphorylation at any one site leads to the inhibition of the complex in vitro. Two pyruvate dehydrogenase phosphatases (PDP1 and PDP2) dephosphorylate the E1α and activate the enzyme. The phosphatases show little or no site specificity. Both the kinases and phosphatases are differentially expressed in tissues. Each of the PDK’s and PDP’s is under transcriptional control in response to different cellular stress events as shown in Figure 2. In addition, the kinases are activated by acetyl coenzyme A, NADH and ATP, meanwhile the availability of pyruvate and ADP leads to their inhibition.

Figure 3 . A schematic of the reactions controlling pyruvate dehydrogenase. As an example of the transcriptional regulation, expression of PDK4 is suppressed under basal conditions in most tissues by maintaining relevant histones in a deacetylated state, but its expression is increased during starvation by glucocorticoids that re-acetylate these histones, particularly in heart, skeletal and other muscle tissues, kidney, and liver. PDK4 is also upregulated by a high fat diet and extended exercise. inhibits PDK4 expression via PI3K signaling that leads to lower histone acetylation. The levels of PDK4 are also regulated to PPAR transcription factors. Importantly, in diabetes caused by either insulin deficiency or insulin insensitivity, the uninhibited PDK4 over-expression prevents glucose oxidation. In contrast, the levels of PDK1 are sensitive to O2 levels and under regulation by the transcription factor HIF-1α. An increase in the level of PDK1 is a key part of the so-called Warburg effect, a switch from oxidative to glycolytic ATP production that characterizes cancer cells.

Mechanism of action

The ylide resonance form of (TPP) begins by attacking the electrophilic ketone of pyruvate. The intermediate β- alkoxide then decarboxylates and the resulting enol is deprotonated on the carbon atom to form a stabilized 1,3-dipole involving a positively charged nitrogen atom of the thiamine heterocycle. This 1,3-dipole undergoes a reductive acetylation with lipoamide-E2. Biochemical and structural data for E1 revealed a mechanism of activation of TPP coenzyme by forming the conserved hydrogen bond with glutamate residue (Glu59 in human E1) and by imposing a V-conformation that brings the N4’ atom of the aminopyrimidine to intramolecular hydrogen bonding with the thiazolium C2 atom. This unique combination of contacts and conformations of TPP leads to formation of the reactive C2- carbanion, eventually. After the TPP decarboxylates pyruvate, the acetyl portion becomes a hydroxyethyl derivative covalently attached to TPP.

Active site of PDH E1 has two catalytic sites, each providing thiamine pyrophosphate (TPP) and magnesium ion as cofactors. The α- subunit binds magnesium ion and pyrophosphate fragment while the β- subunit binds pyrimidine fragment of TPP, forming together a catalytic site at the interface of subunits. The for pyruvate dehydrogenase (image created from PDB: 1NI4 ) holds TPP through metal ligation to a magnesium ion (purple sphere) and through hydrogen bonding to amino acids. While over 20 amino acids can be found in the active site, amino acids Tyr 89, Arg 90, Gly 136, Val 138, Asp 167, Gly 168, Ala 169, Asn, 196, and His 263 actually participate in hydrogen bonding to hold TPP and pyruvate (not shown here) in the active site. The amino acids are shown as wires, and the TPP is in ball and stick form. The active site also aids in the transfer of the acyl on the TPP to a lipoamide waiting on E2.

Pathology associated with PDH

Pyruvate dehydrogenase is an autoantigen recognized in primary biliary cirrhosis. These antibodies appear to recognize oxidized protein that has resulted from inflammatory immune responses. Some of these inflammatory responses could be related to gluten sensitivity as over 50% of the acute liver failure patients in one study exhibited a nonmitochondrial autoantibody against tissue . Other mitochondrial autoantigens include oxoglutarate dehydrogenase and branched-chain alpha-keto acid dehydrogenase complex, which are antigens recognized by antimitochondrial antibodies. Pyruvate dehydrogenase (PDH) deficiency is a congenital degenerative metabolic disease resulting from a mutation of the pyruvate dehydrogenase complex (PDC) located on the . While defects have been identified in all 3 of the complex, the E1-α subunit is predominantly the culprit. Malfunction of the citric acid cycle due to PDH deficiency deprives the body of energy and leads to an abnormal buildup of lactate. PDH deficiency is a common cause of lactic acidosis in newborns and often presents with severe lethargy, poor feeding, tachypnea, and cases of death have occurred.

References

1. PDB: 1ni4 ; Ciszak EM, Korotchkina LG, Dominiak PM, Sidhu S, Patel MS (June 2003). "Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase". J. Biol. Chem. 278 (23): 21240–6. doi:10.1074/jbc.M300339200 . PMID 12651851 . 2. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and See also Informatics at the University of California, San Francisco; Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (October 2004). "UCSF Chimera—a visualization system for exploratory research and analysis". J Comput Chem. 25 (13): 1605–12. doi:10.1002/jcc.20084 . PMID 15264254 . 3. Jaimes, R 3rd (Jul 2015). "Functional response of the isolated, perfused normoxic heart to pyruvate dehydrogenase activation by dichloroacetate and pyruvate" . Pflügers Arch. 468: 131–42. doi:10.1007/s00424- 015-1717-1 . PMC 4701640 . PMID 26142699 . 4. Arjunan P, Nemeria N, Brunskill A, Chandrasekhar K, Sax M, Yan Y, Jordan F, Guest JR, Furey W (2002). "Structure of the pyruvate dehydrogenase multienzyme complex E1 component from at 1.85 A resolution" . Biochemistry. 41 (16): 5213–21. doi:10.1021/bi0118557 . PMID 11955070 . 5. Leung PS, Rossaro L, Davis PA, et al. (2007). "Antimitochondrial antibodies in acute liver failure: Implications for primary biliary cirrhosis" . Hepatology. 46 (5): 1436–42. doi:10.1002/hep.21828 . PMC 3731127 . PMID 17657817 . Pyruvate+Dehydrogenase-E1 at the US National Library of Medicine 6. Pyruvate Dehydrogenase Complex Deficiency at eMedicine 7. Recny MA, Hager LP (1982). "Reconstitution of native Escherichia coli from apoenzyme monomers and FAD". J. Biol. Chem. 257 (21): 12878–86. PMID 6752142 . 8. Abdel-Hamid AM, Attwood MM, Guest JR (2001). "Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli". Microbiology. 147 (Pt 6): 1483–98. doi:10.1099/00221287-147- 6- 1483 . PMID 11390679 . External links Content is available under CC BY-SA 3.0 unless otherwise noted. Medical Subject Headings (MeSH) http://www.brookscole.com/chemistry _d/templates/student_resources/shar ed_resources/animations