Chemistry Department University of Southern Maine
Links To Files Used In Biochemistry Class (CHY 361-363)
Be sure to read the Introduction to the Biochemistry Graphics Gallery to learn how to view these files.
Topic: The Citric Acid Cycle
Lipoamide Arm in H Protein of Glycine Decarboxylase (Convergent Stereo)
The long arm of lipoate-lysine, which is also found in dihydrolipoyl transacetylase, the E2 subunit of pyruvate dehydrogenase.
For graphics from other topics, see Topics List.
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Molecules to Explore Aconitase/Citrate Complex
Aconitase catalyzes the interconversion of citrate and isocitrate, with enzyme-boundcis-aconitate as the intermediate. Here is a model of a site-directed mutant (S642A) aconitase with bound citrate: 1AS9.pdb.
Citrate binds to one iron Fe atom of the Fe4S4cluster at the active site of aconitase. This iron Fe atom has six 6 ligands: three sulfurs S-S-S in the cluster, oxygen O of the C-3 OH group of citrate, one oxygen of the carboxy group on C-3 of citrate, and a water molecule.
C-3 of citrate is not chiral, because it carries two 2 identical carboxymethyl groups, one derived from oxaloacetate, the other from acetyl-CoA. Aconitase distinguishes between these two 2 seemingly identical groups. In the product, isocitrate, the OH group is on the carbon C derived from oxaloacetate, not from acetyl-CoA. The following exercises will help you to see how the enzyme accomplishes this conversion.
Think About It
1. Restrict your view to atoms within 6 or 7 angstroms of citrate, including the iron-sulfur Fe4S4 cluster. Find the iron atom that binds citrate and measure the distance to each of its six 6 ligands.
2. In addition to the iron atom, what residues bind citrate?
3. What additional non-cluster ligand is present on the same iron atom that is bound to citrate? This ligand is one of the substrates of the aconitase reaction.
4. Arrange the view so that you see C-3 of citrate as in a Fisher projection, with the C-3 hydroxyl pointing left and toward you, and the carboxyl on C-3 pointing right and toward you. You will be looking at citrate through the FeS cluster. Above and below C-3 are two 2carboxymethyl groups. The upper one is derived from acetyl-CoA, the lower one from oxaloacetate.
5. Notice that a water molecule (answer to question 3 above) lies above the lower CH2 group, the one derived from oxaloacetate. The CH2 group derived from acetyl-CoA is far away from this water molecule. In the product complex with isocitrate, this water becomes the new OH group on C-2, and the C-3 OH of citrate becomes an Fe-bound water molecule. You might imagine that citrate could bind "upside down" from this orientation, allowing the other CH2 to be the OH acceptor , but note that ARG452, on your right, binds the C-3 carboxyl of citrate. The only way citrate can bind is in the orientation shown in this model, so the CH2 group derived from acetyl-CoA cannot be the acceptor of the new OH group.
RasMol Scripts
Here are some scripts to help you with the questions above. To use them, first start a desktop copy of RasMol. Then click to download the .pdb file. Then, to see each view described, click the appropriate .spt file.
1as9.pdb: Excerpt of the PDB file 1as9, containing only citrate, the FeS cluster, and atoms within 6 angstroms of them.
1as9Hets01.spt: Ball & Stick models of citrate and FeS cluster. Sulfur S atoms are yellow, iron Fe atoms orange.
1as9Hets02.spt: Ball & Stick models of citrate and the FeS cluster, with nearby atoms in wireframe. Note the cysteine sulfurs S (3 of them) that complete the FeS cluster. Also note the Fe closest to citrate. This ion Fe is complexed with a citrate carboxyl and the substrate water molecule. The enzyme removes OH from C-3 and uses the water molecule to replace OH on C-2.
1as9Hets03.spt: Same groups, arranged to show the prochiral C-3 atom of citrate. It is clear from this view that aconitase can distinguish the two 2carboxymethyl groups of citrate.
Malate Dehydrogenase/Malate /NAD+ Complex
Malate dehydrogenase (MDH) catalyzes the reversible oxidation of L-malate to oxalacetate. Click here to download a model of the E. coli MDH with bound NAD+and malate: 1CME.pdb.
Because this complex is catalytically active, it is not possible to determine its structure by crystallography. 1CME is a theoretical model built from a crystallographic model of MDH bound to citrate, which binds in similar fashion to malate. The investigators removed the citrate coordinates from the file, and built a model of NAD+ into the its binding site, based on its position in crystallographic models of MDH/NAD+ complexes. Then they built a malate model into its presumed binding site, based on interactions observed for citrate.
Think About It
· Display the model as a backbone model. Select residues 1-144AA and color them green. Select residues 145-312AA and color them yellow. Then display malate and NAD+ as space-filling models. MDH has two 2 domains. DomainI binds NAD+, and domain II provides the catalytic residues HIS177 and ASP150. Both domains are involved in binding malate.
· Restrict your view to malate and NAD+. What is the distance between C-2 of malate and C-4 of NAD+? During catalysis, a hydride H ion moves between these two 2 carbons C.
· Add atoms within 6 or 7 angstroms Å of malate to the view. What amino acids AA are involved in binding the carboxyls of malate? Which are from domain I and which from domain II?
· Residues HIS177 and ASP150 are essential to catalysis. Add these side chains to the view, and measure the distances between interacting atoms in HIS, ASP, and malate. Note the resemblance of these three groups to the catalytic HIS, ASP, and SER of serine proteases. The position of the C-2 OH of malate is analogous to that of the side-chain OH of SER in serine proteases.
· C-2 of L-malate is chiral. Is its configuration R or S? Remember that there is a hydrogen atom at C-2 that is not shown.
· Imagine that the D-enantiomer of malate were bound at this site, with the carboxyls bound as shown in this model. This would mean that positions of the C-2 OH and the C-2 H atom (not shown) would be swapped. Why can MDH not transfer hydride between NAD andD-malate?
Here's a RasMol script of active-site details. It my help if you had trouble answering the questions above: 1CMEBndg.spt
For SwissPdbViewer Users Only
If you use SwissPdbViewer, you can see what it's like to try to place a substrate model into the active site. Configure your web browser to use SwissPdbViewer for files of MIME type chemical/x-pdb. Then download these two files:
1. MDH.pdb: a model of MDH/NAD+ without malate.
2. Malate.pdb: a model of malate, in the correct conformation for binding.
With these two files loaded into SPdbV, try to place the malate model into the active site of the enzyme. You can move models separately in SPdbV by use of the Control Panel. Each model has a can move button. Click to remove the checkmark from the can move box, and that model will remain motionless while you move other models. It helps to display surface dots on the malate model while trying to fit it into the active site.
Once you have fitted the malate into place, load 1CME.pdb into SPdbV and use Tools: Magic Fit to superimpose 1CME onto MDH. Be sure that MDH is the reference, so that it does not move during the superposition. After superposition, center on malate in 1CME and compare its position to the current position of your malate molecule in the Malate.pdb layer.
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