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Biotin-dependent enzyme

Biotin-dependent enzyme. Pimchai Chaiyen . References: Jitrapakdee 2008, Biochem J. Paul Attwood’s seminar at our department (19 June 2009) Frey & Hegeman 2007, Enzymatic Reaction Mechanisms. Pyruvate Carboxylase - Role In Metabolism. Jitrapakdee and Wallace (1999)

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Biotin-dependent enzyme

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  1. Biotin-dependent enzyme Pimchai Chaiyen References: Jitrapakdee 2008, Biochem J. Paul Attwood’s seminar at our department (19 June 2009) Frey & Hegeman 2007, Enzymatic Reaction Mechanisms

  2. Pyruvate Carboxylase - Role In Metabolism Jitrapakdee and Wallace (1999) Biochem.J. 340, 1-16.

  3. Chemistry of Biotin Enol & ketone Carboxylation & Decarboxylation

  4. Pyruvate Carboxylase - The Reaction Enz-biotin + MgATP + HCO3- MgADP + Pi + Enz-biotin-CO2- Enz-biotin-CO2- + CH3COCO2- Enz-biotin + -O2CCH2COCO2- Attwood (1995) Int. J. Biochem. Cell Biol. 27, 231-249 Question: Role of ATP?

  5. Participation of ATP in catalysis(Biotin carboxylase)

  6. Dependence of Pyruvate Carboxylase on Acetyl CA Organism Activity in the absence of acetyl CoA Ka (M) (% activity with saturating acetyl CoA) Chicken 1a 13b Sheep 25c 15d Yeast (Pyc1) 6e 17e Yeast (Pyc2) 27e 8e Aspergillus niger 100f- Agrobacterium tumefaciens 0f11f Pseudomonas citronellolis 100f- aAttwood et al. (1986) FEBS Lett 203, 191-196 bFrey and Utter (1977) J. Biol. Chem. 252, 51-56 c Ashman et al. (1972) J. Biol. Chem.247, 5818-5824 dBarritt (1985) Pyruvate Carboxylase CRC Press pp 142-176 eJitrapakdee et al. (2007) Int. J. Biochem. Cell. Biol. 39, 1211-1223 f Wallace (1985) Pyruvate Carboxylase CRC Press pp 5-63

  7. Acetyl CoA Prevents Dilution Inactivation Sheep Kidney Pyruvate Carboxylase • 0.4 mg/ml • 0.01 mg/ml + 0.25 mM acetyl CoA •  0.1 mg/ml • 0.05 mg/ml • 0.02 mg/ml x 0.01 mg/ml Question: How can dilution inactivate PC? Ashman et al. (1972) J. Biol. Chem. 247, 5818-5824.

  8. Acetyl CoA Prevents Dilution Inactivation by Preventing Dissociation of the Enzymic Tetramer Time after dilution to 0.025 mg/ml 1 min 40 min 100 min Tetramer Dimer/Monomer - Acetyl CoA Gel Filtration Chromatography Of Chicken PC Following Dilution + Acetyl CoA (0.1 mM) Attwood et al. (1993) Biochem. J. 290, 583-590.

  9. Acetyl CoA Reverses Dilution Inactivation by Inducing Reassociation of the Enzymic Tetramer Specific Activity Aggregates Tetramer Dimer Monomer (% Undiluted) (%) (%) (%) (%) Condition Undiluted 100 2 84 14 0 1 min after dilution 22 12 20 56 12 25 min after dilution 2 20 23 49 8 + acetyl CoA after 90 11 70 14 5 25 min (acetyl CoA added 1 min after dilution) Question: Why monomer cannot function? Attwood and Geeves (2002) Arch. Biochem. Biophys. 401, 63-72.

  10. Acetyl CoA Induces a ‘Compact’ Tetrahedral Form of the Enzymic Tetramer Electron micrographs of pyruvate carboxylase prepared in the presence or absence of acetyl CoA Mayer et al. (1980) Eur. J. Biochem.112, 265-272. - acetyl CoA + acetyl CoA Models of enzymic tetramer Mayer et al. (1980) Eur. J. Biochem.112, 265-272. Partially Flattened Compact Tetrahedron Tetrahedron

  11. Acetyl CoA Induces a ‘Compact’ Tetrahedral Form of the Enzymic Tetramer 20 nm High magnification view of polymer chain 40 nm Formation of polymer chains of tetramers in the presence of avidin and acetyl CoA avidin biotin binding sites active sites Model of polymer chain Model of interaction of avidin with a subunit dimer Johannssen et al. (1983) Eur. J. Biochem. 133, 201-206.

  12. Locus of Action of Acetyl CoA - The Biotin Carboxylation Reaction Acetyl CoA stimulates the rate of carboxylation of free biotin by a biotin-deficient mutant of B. thermodentrificans PC by a factor of 8. Analysis of MgATP cleavage by mutant suggests that a part of the effect of acetyl CoA may be due to enhanced binding of free biotin, which stimulates ATP cleavage. • + acetyl CoA  - acetyl CoA Adina-Zada et al. (2008) Int. J. Biochem. Cell Biol. 40, 1743-17526.

  13. Structure of Rhizobium etli Pyruvate Carboxylase and Acetyl CoA -Binding Site Acetyl CoA binding domain Biotin carboxyl carrier protein domain (contains site of attachment of biotin - K1119) St. Maurice et al. (2007) Science 1076-1079 Question: A working model between the two active sites?

  14. Acetyl CoA binds at the subunit interface

  15. Ethyl CoA (Acetyl CoA analogue) Only Binds to Two of the Four Subunits in the Tetramer St. Maurice et al. (2007) Science 1076-1079 Bottom view showing pair of subunits with no ethyl CoA bound. Note open conformation which does not allow BCCP from one subunit to interact with CT domain of its partner subunit. Half-site reactivity Schematic top view showing pair of subunits with ethyl CoA bound. Note BCCP from one subunit interacts with CT domain of its partner subunit. Top view

  16. Another binding site for biotin, between allosteric and CT

  17. Interesting structural features • Inter-molecular biotin transfer • Half-site reactivity • Multi-subunit and multi-domain cooperation in catalysis

  18. Reaction Mechanism

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