1. How many and which residues of Isocitrate dehydrogenase (IDH) bind with NADP⁺?

a) What are interaction with which atom of NADP+?

b) What is the distance between these atoms?

c) Are there any secondary structural context?

2. Write a proposed mechanism for IDH-catalyzed reaction. Include at least 3 residues that are involved in such a mechanism, and briefly explain what each is doing (e.g. acting as acid/base at a specific step, stabilizing a specific intermediate state, etc..)

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Patrina SchowalterLv2

24 May 2018

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This problem is about the relationship between the structure and function of the enzyme isocitrate dehydrogenase (IDH), which catalyzes the oxidation of isocitrate and subsequent decarboxylation (see Chapter 16 for more details):

isocitrate + NAD(P)+ Â® a-ketoglutarate + CO2 + NAD(P)H + H+
This is one of the reactions in the TCA cycle, and is tightly regulated. One of the ways in which

IDH is regulated in E. coli is by phosphorylation of serine 113, which inhibits it. You will examine the crystal structures in the following PDB files:

PDB ID#	Description
1IDH	Enzyme complexed with NADP+, isocitrate, and Ca2+
2IDH	Enzyme complexed with isocitrate, Mg2+
3IDH	Enzyme phosphorylated at Ser113
4IDH	Mutant enzyme with Ser113 converted to Glu

1) Briefly describe the overall tertiary and quaternary structure of IDH.
(You can use any of the PDB files for this. Approach this as you did in Problem 1.) What kind of domains does it have (e.g. what class)?
What sort of symmetry does the quaternary structure possess?
Briefly describe the dimerization interface â how are the 2 subunits held together? (e.g. Describe how the secondary structural elements come together.)

2) Examine the active site in 1IDH. Which amino acids are part of the sites binding the substrates? Fill out the tables on the next pages for the most important residues you identify for IDHâs function. For the column in which you report distances, indicate which atoms you used to measure the distance (of the amino acid and the substrate or effector).

a)Residues most important for binding isocitrate (âICT1418â)

Residue (name & #)

Atom comes from which part of the residue? Interacts with which atom of isocitrate? Type(s) of interaction (e.g. H-bond, charge-charge)

distance

between atoms (AÌ) & 2Â° structural context

B) Residues most important for binding NADP+ (Note: âd1417â is the NADP+ associated with Chain A, while âNAP1417â is the NADP+ associated with Chain B.)

C) Residues important for binding Ca2+ (which likely is where Mg2+ binds)

3) Write below a proposed mechanism for the IDH-catalyzed reaction. Include at least 3 residues that would be involved in such a mechanism, and briefly explain what each is doing (e.g. acting as acid/base at a specific step, stabilizing a specific intermediate state, etc.)