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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
||Citrate synthase (E.C.
22.214.171.124) (CS) catalyzes the reversible condensation of an acyl group
(from acetil CoA) with oxalacetate to yield citrate and CoA. It is
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
the acetylCoA appears. In this sense, the cell saves acetylCoA as the
not capable of hydrolyzing it efficiently in the open
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
|To start interacting with
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.
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
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 sticks, BindingSite + Ligands
color white, 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)
align 5CSC, 1AL6 zoom show cartoon, 5CSC + 1AL6
color magenta, 1AL6color cyan, 5CSC hide sticks, BindingSitehide lineshide labels
remove chain b
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
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.|
Prepare for 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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