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)Biochem.J. 340, 1-16.

Chemistry of Biotin

Enol & ketoneCarboxylation & Decarboxylation

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?

Participation of ATP in catalysis(Biotin carboxylase)

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?

Acetyl CoA binds at the subunit interface

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

Reaction Mechanism