Title: Citrate Synthase
1Citrate Synthase
- Biol 3100
- April 21, 2008
- Kwabena Sarfo
- Thuy Le
- René Mussell
2Citrate Synthase Background
- Family Acyltransferases
- Small class of enzymes
- Can directly form a carbon-carbon bond
- Does not require metal ion cofactors
- Highly conserved
- e-197
- 6 CSC
- isolated from the inner mitochondrial matrix of
chicken cardiac muscle - structure determined by X-Ray Diffraction
3Citrate Synthase Background
- Location Mitochondrial matrix
- Found in nearly all cells
- capable of oxidative
- metabolism
http//www.becomehealthynow.com/article/bodycell/7
09/3
4Citrate Synthase Function
- Function TCA cycle and cellular energy
- Catalyzes the first step
- acetyl-CoA oxaloacetate H-O ? citrate CoA-SH
5ClustalW Bootstrap Tree
- Compared chicken to
- Cat
- Elephant
- Western clawed frog
- Whale
- Rabbit
- Savannah tsetse fly
- Bootstrap values
- (((Cat,Chicken)26,(Elephant,Western_clawed_frog)
33)12, Whale,(Rabbit,Savannah_tsetse_fly)38)
26
6Clustal Rooted Tree
7PROTPARS Tree Newick Format
- Most parsimonius
- Unrooted tree
- Newick Format
- ((whale,(rabbit,(elephant,((savannah_t,western_cl)
,chicken)))),cat)
8ClustalW
- Multiple Sequence Alignment (MSA)
- Consensus Key
9Box Shade
- Number of amino acids varies by species
- Highly conserved regions may be necessary for
function
Key Completely Conserved Residues
red Identical Residues green Similar Residues
blue Different Residues yellow
10Primary Structure
- Two chains (A B)
- 437 amino acids
- (per chain)
- 100 identity
11Predicted Secondary Structure
- PELE (JOI)
- Chooses if the sequence is more likely to be in a
helix or a sheet - 10 possible a-helices
- 4 possible b-sheets
- CHOFAS
- Predicts all the sequences that show a
possibility to form a certain secondary structure - Tends to be more accurate
- 18 possible a-helices
- 15 possible b-sheets
12Predicted Secondary Structure
- 2o structure corresponding to primary sequence
13Actual Secondary Structure
- 20 a-helices (per subunit)
- 2 anti-parallel b-strands
- (per subunit)
- Random coil
14Supersecondary, Tertiary, and Quaternary Structure
- Supersecondary
- Orthogonal bundle of a-helices
- Tertiary
- Globular
- Quaternary
- Dimer of two identical subunits
- Subunits may interact with the membrane or other
membrane-bound proteins
15Predicted Transmembrane Regions
- Hydropathy
- 1 possible TM region
- TMAP
- 1 possible TM region from aa392-416
- TMHMM
- No predicted regions
16Function
- Conversion of oxaloacetate and acetyl-CoA into
citrate - The binding site is located between the cleft of
the two subunits
17Important Residues
Cofactor trifluoroacetonyl-coa Ligand
oxaloacetate?citrate
18Mechanism
- Oxaloacetate enters open binding site
- Subunits close partially to form binding site for
acetyl-CoA - Subunits close completely around forming
intermediate - Prevent unwanted hydrolysis of thiol ester
19 Mechanism, cont.
- Deprotonated Asp375 residue removes a methyl
proton from acetyl-CoA - Internal stabilization results in an enol
intermediate interacting with the deprotonated
amine of His274 residue
http//bcs.whfreeman.com/lehninger/ Mechanism
Animations, Chapter 16
20 Mechanism, cont.
- Deprotonated His274 regains its proton
- Electrons from the enol CC attack the
keto-carbon of oxaloacetate - His320 becomes deprotonated
- Citroyl-CoA intermediate forms
http//bcs.whfreeman.com/lehninger/ Mechanism
Animations, Chapter 16
21 Mechanism, cont.
- The thioester is hydrolyzed
- CoA-SH dissociates
- Subunits open and citrate leaves
http//bcs.whfreeman.com/lehninger/ Mechanism
Animations, Chapter 16
22Computational Techniques
- Galileo
- Entrez
- PubMed
- Protein Explorer
- PDB Viewer
- Cn3D
- Biology Workbench
- BLASTP
- PELE
- CHOFAS
- Hydropathy
- TMAP
- TMHMM
- CLUSTALW
- PROTPARS
- MSA/Box Shade
23References
- Karpusas M, Branchaud B, Remington SJ. Proposed
Mechanism for the Condensation Reaction of
Citrate Synthase 1.9-A Structure of the Ternary
Complex with Oxaloacetatye and Carboxymethl
Coenzyme A. Biochemistry. 1990. 29, 2213-2219.
- Liao DI, Karpusas M, Remington SJ. Crystal
Structure of an Open Conformation of Citrate
Synthase from Chicken Heart at 2.8-A Resolution.
Biochemistry. 1991, 30, 6031-6036. - Stanislaus F, Souza D, Srere P. Binding of
Citrate Synthase to Mitochondrial Inner
Membranes. J. Biol. Chem. 1983.
258(8)4706-4709. - Usher KC, Remington SJ, Martin DP, Drueckhammer
DG. A very short hydrogen bond provides only
moderate stabbilization of an enzyme-inhibitor
complex of citrate synthase. Biochemistry. 1994
Jun 28 33(25)7753-9 - Wiegand G, Remington S, Deisenhofer J, Huber R.
Crystal structure analysis and molecular model of
a complex of citrate synthase with oxaloacetate
and S-acetonyl-coenzyme A. J Mol Biol. 1984 Mar
25 174(1)205-19.
24Questions?