Graphics Gallery

Gale Rhodes
Chemistry Department
University of Southern Maine

Revised 2006/08/02

Learn how to use Swiss-PdbViewer. Work through sections 1-4 of the Swiss-PdbViewer Tutorial.

Topic: Citric Acid Cycle

Lipoamide Arm in H Protein of Glycine Decarboxylase (Convergent Stereo)

Molecules to Explore

Aconitase/Citrate Complex

Aconitase catalyzes the interconversion of citrate and isocitrate, with enzyme-bound cis-aconitate as the intermediate. Here is a model of a site-directed mutant (S642A) aconitase with bound fluorocitrate, an unreactive substrate analog: 1BOK.pdb. The fluoride atom could not be seen in the electron density, so it was modeled as citrate.

Citrate binds to one iron atom of the Fe₄S₄ cluster at the active site of aconitase. This iron atom has six ligands: three sulfurs in the cluster, oxygen 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 identical carboxymethyl groups, one derived from oxaloacetate, the other from acetyl-CoA. Aconitase distinguishes between these two seemingly identical groups. In the product, isocitrate, the OH group is on the carbon 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 4 or 5 angstroms of flurocitrate (FLC756), including the iron-sulfur cluster (FS4757). Find the iron atom that binds citrate and measure the distance to each of its six 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 carboxymethyl 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.

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 1-144 and color them green. Select residues 145-312 and color them yellow. Then display malate and NAD⁺ as space-filling models. MDH has two domains. Domain I 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 ion moves between these two carbons.
• Add atoms within 6 or 7 angstroms of malate to the view. What amino acids 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 and D-malate?

Using SwissPdbViewer, you can see what it's like to try to place a substrate model into the active site. Make sure that Netscape is using 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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