This problem is about the relationship between the structure and function of the

Question

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)+ ® 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 – 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 IDH’s 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 (“ICT1418”)

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 (A) & 2° structural context

B) Residues most important for binding NADP+ (Note: “d1417” is the NADP+ associated with Chain A, while “NAP1417” 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.)

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

Explanation / Answer

Isocitrate dehydrogenase(IDH) is a very important enzyme of the TCA cycle which is take place in the matrix of mitrocondria. IDH is responsible for catalyzing the reversible conversation of isocitrate to Alpha-ketoglutorate and Co2 into two steps.1) firt step involves the oxidation of the intermediate Oxalosuccinate,2) second step looses the beta-carboxylate of the oxalosuccinate intermediate as carbon dioxide leaving the alpha-ketoglutorate. while the catalytic reaction of isocitrate to alpha-ketogulatarate either NADH or NADPH is produced along with carbon dioxide.

IDHs is homodimer or hetrodimers of 70-416 residues with 40-50 kDa moleculer weight. the structure of IDH contain fourteen alpha helix and eighteen beta sheet in parellel and anti parallel are found in the cneter of the structure where alpha helix is found are found all over the structure.

IDH(1IDH) contains two domains the small domain contains the 19 residues where larger domain have the 75 residues, the tertiary structure of IDH is dependent on IDH hetrodimers intrecting with one another to form a hetrotetramers. Residual structure obstructs the binding site for AMP. When citrate binds to IDH1 by the help of Thr-241, the helix unfolds and truns into an extended loop, allowing residues move into the space previously occupied by the helix, opening the AMP-binding site.

AMP binding is mainly dependent on  citrate binding at the IDH1 binding site, the binding of NAD+ in IDH2 is dependent on prior isocitrate binding at the catalytic site, which in turn is dependent on prior Mg2+ binding. NADP+ is binds to the active site of the interdomain cleft, the interations with isocitrate and magnesium help to align the nicotinamide ring (present in the chain B) so a hydric transfer can happen. Magnesium have a important role in stablization of the intermediate by forming an octahedral arrangment of isocitrate.

IDH residues forms disulphide bonds when the citrate is bind to the IDH this disulphide bonds are reduced and the hydrogen bonds in beta- barrel are also broken and then the IDH residues adopt the structural or binding conformation.

for the further information about the domain , interactive site, and 3D model the fasta sequence of the 1IDH has been provided which is given below , it can be all done by the SWISSMODEL and PDB databank.

ChainA:IVCHTTATSPISAVTCPPGENLCYRKMWCDAFCSSRGKVVELGCAATCPSKKPYEEVTCCSTDKCNPHPKQRPG ChainB:YRGWKHWVYYTCCPDTPYX

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