The following questions are based on material presented in EMS 9A and 9B.
 

EMS 9A

K1
1. a) Examine the structure of citrate synthetase.  How many amino acids does citrate synthetase have? Describe the structure of citrate synthetase in terms of its secondary elements.
b) What is are the substrates for citrate synthetase?
c)  A conformational change occurs as the substrates  bind to  citrate synthetase. Describe this conformational change.

K2
2. a) Identify the amino acids that make hydrogen bonds with oxaloacetate in citrate synthetase.  Are these amino acids acting as hydrogen bond donors or acceptors?
b) As acetyl CoA binds, the conformational change gives rise to additional hydrogen bonds- how many additional hydrogen bonds are formed between substrate and enzyme?   What amino acids are involved in making the additional hydrogen bonds?
c) As CoA is released, the enzyme active site returns to the open conformation and the number of hydrogen bonds to the product decreases.  How many hydrogen bonds are there after CoA is released?  What amino acids are involved?  Why is a decrease in the number of hydrogen bonds after product formation advantageous?

K3
3, a)  How many amino acids does aconitase have.  Describe the secondary structure of aconitase.
b) Aconitase has a [4Fe-4S] cluster at its active site which is held in place by coordination of 3 of its Fe's to 3 cysteine residues.  Which three cysteine residues are coordinated to the Fe's of the cubane?   How far are the cysteine residues from the Fe's?
c) Like citrate synthetase, the substrate makes hydrogen bonds with amino acids in the active site. Identify possible hydrogen bonds. How many hydrogen bonds are there and how many of them are between the COO- and OH groups on C-3?
d) The cubane also interacts with the substrate, what iron atom from the cubane forms bonds with the cluster?  To what atoms does the substrate bind?
e) The interactions between the cubane and substrate  are important, why?
f) Identify the hydrogen bonds that form between the product (isocitrate) and the enzyme.
g) What happens to the cubane as isocitrate forms?
h)   Describe how the active site geometry of aconitase assures that only one chiral product is formed from the  prochiral substrate.
 

EMS 9B

K5
4, a) Examine the structure of isocitrate dehydrogenase.  How many amino  acids does this enzyme  have?  Describe what secondary structural elements are present. What cofactors are required for this enzyme's activity?

K6
5, a)Identify the nature of the hydrogen bonds between the substrate (isocitrate) and isocitrate dehydrogenase.
b) A cation plays an important role in the enzyme,  what is the cation and what amino acids ligate to the cation?  What else ligates to the cation?  In what  geometry is the cation bound?  How close is the cation to the reactive hydroxyl group on the isocitrate?
c) What happens to the bonding patterns of the  isocitrate and the cation as the NADPH binds?
d) As a-ketoglutarate is formed, what happens to the bonding patterns of the cation and product (compared to when the substrate is bound)?

K8
6. a) Examine the structure of malate dehydrogenase.  How many amino  acids does this enzyme  have?  Describe what secondary structural elements are present.
b) Examine the structure of malate dehydrogenase with NADH and malate bound.  Where is the malate with respect to the solvent? Where is the NADH reactive ring?
c) How many hydrogen bonds does the enzyme form with malate.  What amino acids are involved and what portions of the malate are involved in forming the H-bonds?
d) Examine the structure of the enzyme with oxaloacetate bound. Do the same amino acids make H-bonds with oxaloacetate as with malate? Are there more of fewer H-bonds between oxaloacetate and the enzyme compared to malate bound?
e) His -177 plays an important role in the conversion of malate into oxaloacetate. What is His-177's role?  What is the distance between the His-177, the C-4 atom of NAD+, and the reactive OH (o2) of  malate?