Citrate Synthase

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Citrate Synthase

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

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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

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Citrate Synthase Background

• Location Mitochondrial matrix
• Found in nearly all cells
• capable of oxidative
• metabolism

http//www.becomehealthynow.com/article/bodycell/7
09/3
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Citrate Synthase Function

• Function TCA cycle and cellular energy
• Catalyzes the first step
• acetyl-CoA oxaloacetate H-O ? citrate CoA-SH

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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)

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Clustal Rooted Tree
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PROTPARS Tree Newick Format

• Most parsimonius
• Unrooted tree
• Newick Format
• ((whale,(rabbit,(elephant,((savannah_t,western_cl)
  ,chicken)))),cat)

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ClustalW

• Multiple Sequence Alignment (MSA)
• Consensus Key

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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
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Primary Structure

• Two chains (A B)
• 437 amino acids
• (per chain)
• 100 identity

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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

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Predicted Secondary Structure

• 2o structure corresponding to primary sequence

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Actual Secondary Structure

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

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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

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Predicted Transmembrane Regions

• Hydropathy
• 1 possible TM region
• TMAP
• 1 possible TM region from aa392-416

• TMHMM
• No predicted regions

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Function

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

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Important Residues

• His-274
• His-320
• Asp-375

Cofactor trifluoroacetonyl-coa Ligand
oxaloacetate?citrate
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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

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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
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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
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Mechanism, cont.

• The thioester is hydrolyzed
• CoA-SH dissociates
• Subunits open and citrate leaves

http//bcs.whfreeman.com/lehninger/ Mechanism
Animations, Chapter 16
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Computational Techniques

• Galileo
• Entrez
• PubMed
• Protein Explorer
• PDB Viewer
• Cn3D

• Biology Workbench
• BLASTP
• PELE
• CHOFAS
• Hydropathy
• TMAP
• TMHMM
• CLUSTALW
• PROTPARS
• MSA/Box Shade

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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.

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Questions?