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Citrate Synthase

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Small class of enzymes. Can directly form ... isolated from the inner mitochondrial matrix of chicken cardiac muscle ... Savannah tsetse fly. Bootstrap values: ... – PowerPoint PPT presentation

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Title: Citrate Synthase


1
Citrate Synthase
  • Biol 3100
  • April 21, 2008
  • Kwabena Sarfo
  • Thuy Le
  • René Mussell

2
Citrate 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

3
Citrate Synthase Background
  • Location Mitochondrial matrix
  • Found in nearly all cells
  • capable of oxidative
  • metabolism

http//www.becomehealthynow.com/article/bodycell/7
09/3
4
Citrate Synthase Function
  • Function TCA cycle and cellular energy
  • Catalyzes the first step
  • acetyl-CoA oxaloacetate H-O ? citrate CoA-SH

5
ClustalW 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
6
Clustal Rooted Tree
7
PROTPARS Tree Newick Format
  • Most parsimonius
  • Unrooted tree
  • Newick Format
  • ((whale,(rabbit,(elephant,((savannah_t,western_cl)
    ,chicken)))),cat)

8
ClustalW
  • Multiple Sequence Alignment (MSA)
  • Consensus Key

9
Box 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
10
Primary Structure
  • Two chains (A B)
  • 437 amino acids
  • (per chain)
  • 100 identity

11
Predicted 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

12
Predicted Secondary Structure
  • 2o structure corresponding to primary sequence

13
Actual Secondary Structure
  • 20 a-helices (per subunit)
  • 2 anti-parallel b-strands
  • (per subunit)
  • Random coil

14
Supersecondary, 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

15
Predicted Transmembrane Regions
  • Hydropathy
  • 1 possible TM region
  • TMAP
  • 1 possible TM region from aa392-416
  • TMHMM
  • No predicted regions

16
Function
  • Conversion of oxaloacetate and acetyl-CoA into
    citrate
  • The binding site is located between the cleft of
    the two subunits

17
Important Residues
  • His-274
  • His-320
  • Asp-375

Cofactor trifluoroacetonyl-coa Ligand
oxaloacetate?citrate
18
Mechanism
  • 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
22
Computational Techniques
  • Galileo
  • Entrez
  • PubMed
  • Protein Explorer
  • PDB Viewer
  • Cn3D
  • Biology Workbench
  • BLASTP
  • PELE
  • CHOFAS
  • Hydropathy
  • TMAP
  • TMHMM
  • CLUSTALW
  • PROTPARS
  • MSA/Box Shade

23
References
  • 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.

24
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