User:Fsegarra

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Propionyl Coenzyme-A Carboxylase

Enzyme Structure[edit]

Propionyl-CoA Carboxylase (PCC) is a 750 kDa alpha(6)-beta(6)-dodecamer. (Only approximately 540 kDa is native enzyme. [1] ) The alpha subunits are arranged as monomers, decorating the central beta-6 hexameric core. Said core is oriented as a short cylinder with a hole along its axis.

The alpha subunit of PCC contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains. A domain known as the BT domain is also located on the alpha subunit and is essential for interactions with the beta subunit. The 8-stranded anti-parallel beta barrel fold of this domain is particularly interesting. The beta subunit contains the carboxyltransferase (CT) activity.

The BC and CT sites are approximately 55 A apart, indicative of the entire BCCP domain translocating during catalysis of the carboxylation of propionyl-CoA. [2] This provides clear evidence of crucial dimeric interaction between alpha and beta subunits.[3]

The biotin-binding pocket of PCC is hydrophobic and highly conserved. Biotin and propionyl-CoA bind perpendicular to each other in the oxyanion hole containing active site. The native enzyme to biotin ratio has been determined to be one mole native enzyme to 4 moles biotin. [4] The N1 of biotin is thought to be the active site base.[5]

Site-directed mutagenesis at D422 showed a change in the substrate specificity of the propionyl-CoA binding site, thus indicating this residue’s importance in PCC’s catalytic activity. [6] In 1979, inhibition by phenylglyoxal determined that a phosphate group from either propionyl-CoA or ATP reacts with an essential arginine residue in the active site during catalysis.[7] Later (2004), it was suggested that Arginine-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin. [8]

The KM values for ATP, propionyl-CoA, and bicarbonate has been determined to be 0.08 mM, 0.29 mM, and 3.0 mM respectively. The isoelectric point falls at pH 5.5. PCC’s structural integrity is conserved over the temperature range of -50 to 37 degrees Celsius and the pH range of 6.2 to 8.8. Optimum pH was shown to be between 7.2 and 8.8 without biotin bound.[9] With biotin, optimum pH is 8.0-8.5.[10]

Crystallography

Enzyme Mechanism[edit]

The normal catalytic reaction pathway involves a carbanion intermediate and does not proceed through a concerted process.[11] The diagram shown below is accurate with two minor exceptions: (1) the first step shown should be split in two, with the phosphorylation of bicarbonate happening before the biotinyl-enzyme attacks, and (2) the resonance structure given for propionyl-CoA is not the major resonance structure.

Mechanism Diagram

The reaction has been shown to be slightly reversible at low propionyl-CoA flux.[12]


Pathology

“”

PCC activity is the most sensitive indicator of biotin status tested to date. In future pregnancy studies, the use of lymphocyte PCC activity data should prove valuable in assessment of biotin status.[13] 

Graphs from study

Regulation[edit]

Of Propionyl-CoA Carboxylase[edit]

a. Carbamazepine (antiepileptic drug): significantly lowers enzyme levels in the liver[14]

b. ‘‘E. coli’’ chaperonin proteins groES and groEL: essential for folding and assembly of human PCC heteromeric subunits[15]

c. Bicarbonate: negative cooperativity[16]

d. Mg2+ and MgATP2-: allosteric activation[17]

By Propionyl-CoA Carboxylase[edit]

a. 6-Deoxyerythronolide B: decrease in PCC levels lead to increased production [18] b. Glucokinase in pancreatic beta cells: precursor of beta-PCC shown to decrease KM and increase Vmax; activation [19]

  1. ^ Kalousek F, Darigo MD, Rosenberg LE. Isolation and characterization of propionyl-CoA carboxylase from normal human liver. Evidence for a protomeric tetramer of nonidentical subunits. J Biol Chem. 1980 Jan 10;255(1):60-5. PMID 6765947
  2. ^ Huang CS, Sadre-Bazzaz K, Shen Y, Deng B, Zhou ZH, Tong L. Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase. Nature. 2010 Aug 19;466(7309):1001-5. PMID 20725044; PMC 2925307
  3. ^ Diacovich L, Mitchell DL, Pham H, Gago G, Melgar MM, Khosla C, Gramajo H, Tsai SC. Crystal structure of the beta-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity. Biochemistry. 2004 Nov 9;43(44):14027-36. PMID 15518551
  4. ^ Kalousek F, Darigo MD, Rosenberg LE. Isolation and characterization of propionyl-CoA carboxylase from normal human liver. Evidence for a protomeric tetramer of nonidentical subunits. J Biol Chem. 1980 Jan 10;255(1):60-5. PMID 6765947
  5. ^ Diacovich L, Mitchell DL, Pham H, Gago G, Melgar MM, Khosla C, Gramajo H, Tsai SC. Crystal structure of the beta-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity. Biochemistry. 2004 Nov 9;43(44):14027-36. PMID 15518551
  6. ^ Arabolaza A, Shillito ME, Lin TW, Diacovich L, Melgar M, Pham H, Amick D, Gramajo H, Tsai SC. Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor. Biochemistry. 2010 Aug 31;49(34):7367-76. PMID 20690600; PMC 2927733
  7. ^ Wolf B, Kalousek F, Rosenberg LE. Essential arginine residues in the active sites of propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase. Enzyme. 1979;24(5):302-6. PMID 510274
  8. ^ Sloane V, Waldrop GL. Kinetic characterization of mutations found in propionic acidemia and methylcrotonylglycinuria: evidence for cooperativity in biotin carboxylase. J Biol Chem. 2004 Apr 16;279(16):15772-8. Epub 2004 Feb 11. PMID 14960587
  9. ^ Kalousek F, Darigo MD, Rosenberg LE. Isolation and characterization of propionyl-CoA carboxylase from normal human liver. Evidence for a protomeric tetramer of nonidentical subunits. J Biol Chem. 1980 Jan 10;255(1):60-5. PMID 6765947
  10. ^ Hsia YE, Scully KJ, Rosenberg LE. Human propionyl CoA carboxylase: some properties of the partially purified enzyme in fibroblasts from controls and patients with propionic acidemia. Pediatr Res. 1979 Jun;13(6):746-51. PMID 481943
  11. ^ Stubbe J, Fish S, Abeles RH. Are carboxylations involving biotin concerted or nonconcerted? J Biol Chem. 1980 Jan 10;255(1):236-42. PMID 7350155
  12. ^ Reszko AE, Kasumov T, Pierce BA, David F, Hoppel CL, Stanley WC, Des Rosiers C, Brunengraber H. Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver. J Biol Chem. 2003 Sep 12;278(37):34959-65. Epub 2003 Jun 24. PMID 12824185
  13. ^ Stratton SL, Bogusiewicz A, Mock MM, Mock NI, Wells AM, Mock DM. Lymphocyte propionyl-CoA carboxylase and its activation by biotin are sensitive indicators of marginal biotin deficiency in humans. Am J Clin Nutr. 2006 Aug;84(2):384-8. PMID 16895887; PMC 1539098
  14. ^ Rathman SC, Eisenschenk S, McMahon RJ. The abundance and function of biotin-dependent enzymes are reduced in rats chronically administered carbamazepine. J Nutr. 2002 Nov;132(11):3405-10. PMID 12421859
  15. ^ Kelson TL, Ohura T, Kraus JP. Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli. Hum Mol Genet. 1996 Mar;5(3):331-7. PMID 8852656
  16. ^ Sloane V, Waldrop GL. Kinetic characterization of mutations found in propionic acidemia and methylcrotonylglycinuria: evidence for cooperativity in biotin carboxylase. J Biol Chem. 2004 Apr 16;279(16):15772-8. Epub 2004 Feb 11. PMID 14960587
  17. ^ McKeon C, Wolf B. Magnesium and magnesium adenosine triphosphate activation of human propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase. Enzyme. 1982;28(1):76-81. PMID 6981505
  18. ^ Zhang H, Boghigian BA, Pfeifer BA. Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli. Biotechnol Bioeng. 2010 Feb 15;105(3):567-73. PMID 19806677
  19. ^ Shiraishi A, Yamada Y, Tsuura Y, Fijimoto S, Tsukiyama K, Mukai E, Toyoda Y, Miwa I, Seino Y. A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity. J Biol Chem. 2001 Jan 26;276(4):2325-8. Epub 2000 Nov 20. PMID 11085976
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