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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 - 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?
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 0f 11f
Pseudomonas citronellolis 100f -
aAttwood et al. (1986) FEBS Lett 203, 191-196bFrey and Utter (1977) J. Biol. Chem. 252, 51-56c Ashman et al. (1972) J. Biol. Chem. 247, 5818-5824dBarritt (1985) Pyruvate Carboxylase CRC Press pp 142-176eJitrapakdee et al. (2007) Int. J. Biochem. Cell. Biol. 39, 1211-1223f Wallace (1985) Pyruvate Carboxylase CRC Press pp 5-63
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
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?
Acetyl CoA Prevents Dilution Inactivation byPreventing Dissociation of the Enzymic Tetramer
1 min 40 min 100 minTime after dilutionto 0.025 mg/ml
Tetramer
Dimer/Monomer
- Acetyl CoA
+ Acetyl CoA(0.1 mM)
Gel Filtration ChromatographyOf Chicken PC Following Dilution
Attwood et al. (1993)Biochem. J. 290, 583-590.
Acetyl CoA Reverses Dilution Inactivation byInducing 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 14 525 min (acetyl CoA added1 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?
Acetyl CoA Induces a ‘Compact’ Tetrahedral Formof the Enzymic Tetramer
- acetyl CoA + acetyl CoA
Electron micrographsof pyruvate carboxylaseprepared in the presenceor 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
Acetyl CoA Induces a ‘Compact’ Tetrahedral Formof 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.
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.
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?
Ethyl CoA (Acetyl CoA analogue) Only Binds toTwo of the Four Subunits in the Tetramer
Schematic top view showingpair of subunits with ethyl CoA bound. Note BCCP from one subunit interacts with CT domain of its partner subunit.
Top viewBottom view showing pair ofsubunits 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
Another binding site for biotin, between allosteric and CT
Interesting structural features
• Inter-molecular biotin transfer• Half-site reactivity• Multi-subunit and multi-domain cooperation in
catalysis