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Enzymatic catalysis: citrate synthase

Citrate synthase is the first enzyme in the citric acid cycle (tca cycle). The tca cycle is crucial for aerobic oxidation of glucose and other carbon sources to carbon dioxide. The following concepts should be exemplified by citrate synthase:
  • the catalytic activity of enzymes;
  • quantitative enzyme kinetic calculations;  the determination of affinities (Km values), macroscopic and molecular activities (maximal velocities, specific activities, turnover numbers) of enzymes; substrate specificities;
  • active sites for substrate binding; dynamic movement of protein domains upon binding (“induced fit”); catalysis by transition state stabilization;
  • allosteric regulation of enzymatic activity; regulation by reversible posttranslational modifications; enzyme precursors (proproteins, preproproteins) and their activation by specific cleavage;
  • aerobic glucose catabolism and energy transduction (glycolysis, tca cycle, electron transport chain, ATP synthesis).
Proposed chapters are NC 6, 14.1, 16, 19 or VV

Reset Pymol
citrate synthase
PDB: 1AL6
Citrate synthase (E.C. 2.3.3.1) (CS) catalyzes the reversible condensation of an acyl group (from acetil CoA) with oxalacetate to yield citrate and CoA. It is critical in the Krebs cycle, and of central importance in aerobic cells. It is localized in the inner membrane-matrix of the mitochondria. The enzyme exists in three different stable conformations, one "open" and two "closed". The reaction it catalyzes is particularly interesting because only when oxalacetate binds, which causes the change from "open" to "close",  the binding site for the acetylCoA appears. In this sense, the cell saves acetylCoA as the enzyme is not capable of hydrolyzing it efficiently in the open conformation.

In this module we will study 3 different structures:
5CSC: CS free (open structure)
1AL6: CS complexed with N-OH-amido-CoA and oxalacetate
6CSC: CS complexed with tri-F-acetonyl-CoA and citrate
Launch Pymol


Load 1al6
fetch 1AL6

Show cartoon
show cartoon

To start interacting with the structure, launch pymol and wait until the word "Ready" appears.
Click to load the structure or use the "PDB Loader Service" (under "Plugins" menu) and enter 1AL6 as PDB code.
The first step is to get a feeling of the general structure of the protein. For that, a simplified cartoon representation is the most informative. From the S(how) menu, select cartoon. Rotate the structure to get an overview.
CS is an homodimeric protein. Here we are looking at one monomer. The structure is mostly alpha helical. The 20 helices are disposed in kind of layers. There is also a small portion of an antiparalell beta-sheet.

Show surface
hide lines
show surface

Remove waters remove resn HOH+WAT  Create "binding site" and ligands object
create BindingSite, byres (resn HAX+OAA around 8)
create Ligands, resn HAX+OAA
Let's now identify the binding site. For that the easiest way is to create a new object, comprising the residues in the binding site, i.e., those around the ligand. To find the ligand, we switch to surface representation ("S"how-> surface). For clarity, we also remove crystallographic water molecules ("A"ction->remove waters).
Select the two ligands by left-clicking on any atom of each one.
From the "A"ctions menu of the selection select "modify" -> "around" -> "residues within 8 Å", and this will capture the binding site.  Create an new object also, and rename it to "BindingSite" ("A"ctions->"create object"; from the newly created object:"A"ctions->"rename object"). Create the ligands also as separate objects.

Show binding site
hide all
show sticks, BindingSite + Ligands
color white, BindingSite
zoom BindingSite

Show set1 and set2
select set1, resi 274+320+375
color green, set1
select set2, resi 238+329+401
color red, set2label name ca and byres set1 + set2, "%s - %s"%(resn, resi)
To better explore the binding site, hide the rest of the protein (from the "all" menu, "H"ide->everything) and show the binding site in stick representation (from "BindingSite" menu, "S"how->sticks). The oxalacetate binds mainly to His 274, His 320 and Asp 375 (set1) and also to His 238, Arg 329 and Arg 401 (set2).

Let's highlight these residues and color them differently (red and green). Observe how the side chains of these residues are oriented towards the oxalacetate. Show labels for the residues (from the "L"abel menu of the new selections -> residues)

Load 5csc
fetch 5CSC


Align structures
align 5CSC, 1AL6 zoom show cartoon, 5CSC + 1AL6
color magenta, 1AL6color cyan, 5CSC hide sticks, BindingSitehide lineshide labels
Remove monomer
remove chain b
zoom
Let's now compare with the unbound structure ("open") to appreciate the conformational change upon binding. First we have to load the structure, using the PDB loader service from the plugins menu. The PDB code is 5CSC.
In this case, we have the dimeric structure. First, we align both structures. Second, we remove the extra chain from the new structure. Go with the mouse over it. Right click, "chain"->"remove atoms". For clarity, we choose a cartoon representation of the new structure. Let's also color in magenta the closed structure and in cyan the open one. If we rotate the molecules for an overview, we observe that both structures align quite well in general except for one small area in the vecinity of the binding site.
Explore open binding siteselect set1_open, resi 274+320+375 and 5CSC

select set2_open, resi 238+329+401 and 5CSC
show sticks, set1 + set2 + set1_open + set2_open + set2
If we look at how the residues that constitute the binding site of the oxalate (called before "set1" and "set2") are disposed in the unbound structure compared to the bound one, we observe that the set2 almost does not change: the binding site for the oxalate is preformed. It is the stabilization of the set1 due to the appearance of the oxalate in the binding site that is pivotal to the conformational change, through which the binding site for the acetylCoA appear.
Load 6csc
Prepare for movie
Create movie
Play movie
Stop movie
Finally, load also the structure with the citrate (6csc) after the reaction and compare the differences with the previous two structures.

Provided the three structures have been loaded, the left link will create a short movie for you. First, the open structure without ligands. Second, the open structure plus the oxalate. Third, the closed structure plus the oxalate, then the closed structure with both ligands and, finally, the closed structure with the citrate. 

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