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 + HCO 3 - MgADP + Pi + Enz-biotin-CO 2 - Enz-biotin-CO 2 - + CH 3 COCO 2 - Enz-biotin + - O 2 CCH 2 COCO 2 - 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 K a (  M) (% activity with saturating acetyl CoA) Chicken 1 a 13 b Sheep25 c 15 d Yeast (Pyc1) 6 e 17 e Yeast (Pyc2)27 e 8 e Aspergillus niger100 f - Agrobacterium tumefaciens0 f 11 f Pseudomonas citronellolis100 f - a Attwood et al. (1986) FEBS Lett 203, 191-196 b Frey and Utter (1977) J. Biol. Chem. 252, 51-56 c Ashman et al. (1972) J. Biol. Chem. 247, 5818-5824 d Barritt (1985) Pyruvate Carboxylase CRC Press pp 142-176 e Jitrapakdee et al. (2007) Int. J. Biochem. Cell. Biol. 39, 1211-1223 f Wallace (1985) Pyruvate Carboxylase CRC Press pp 5-63

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

8. Acetyl CoA Prevents Dilution Inactivation by Preventing Dissociation of the Enzymic Tetramer 1 min40 min100 min Time after dilution to 0.025 mg/ml Tetramer Dimer/Monomer - Acetyl CoA + Acetyl CoA (0.1 mM) Gel Filtration Chromatography Of Chicken PC Following Dilution Attwood et al. (1993) Biochem. J. 290, 583-590.

9. Acetyl CoA Reverses Dilution Inactivation by Inducing Reassociation of the Enzymic Tetramer 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) Specific Activity Aggregates Tetramer Dimer Monomer (% Undiluted) (%) (%) (%) (%) Condition Attwood and Geeves (2002) Arch. Biochem. Biophys. 401, 63-72. Question: Why monomer cannot function?

10. Acetyl CoA Induces a ‘Compact’ Tetrahedral Form of the Enzymic Tetramer - acetyl CoA+ acetyl CoA Electron micrographs of pyruvate carboxylase prepared in the presence or absence of acetyl CoA Mayer et al. (1980) Eur. J. Biochem. 112, 265-272. 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 Formation of polymer chains of tetramers in the presence of avidin and acetyl CoA 20 nm High magnification view of polymer chain avidin Model of polymer chain Model of interaction of avidin with a subunit dimer biotin binding sites 40 nm active sites 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. n + 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 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 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 St. Maurice et al. (2007) Science 1076-1079

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