The CAS number for this type of the enzyme is [9028-48-2].
Isocitrate + NADP+ Mg2+(metal ion) --> alpha-ketoglutarate + NADPH+H+ + CO2
The second box is Step 1 which is the oxidation of the alpha-C (C#2). Oxidation is the first step that isocitrate goes through. In this process, the alcohol group off the alpha-carbon (C#2) is deprotonated and the electrons flow to the alpha-C forming a ketone group and removing a hydride off C#2 using NAD+/NADP+ as an electron accepting cofactor. The oxidation of the alpha-C allows for a position where electrons (in the next step) will be coming down from the carboxyl group and pushing the electrons (making the double bonded oxygen) back up on the oxygen or grabbing a nearby proton off a nearby Lysine amino acid.
The third box is Step 2 which is the decarboxylation of oxalosuccinate. In this step, the carboxyl group oxygen is deprotonated by a nearby Tyrosine amino acid and those electrons flow down to carbon 2. Carbon dioxide leaves the beta Carbon of isocitrate as a leaving group with the electrons flowing to the ketone oxygen off the alpha-C placing a negative charge on the oxygen of the alpha-C and forming an alpha-beta unsaturated double bond between carbons 2 and 3. The lone pair on the alpha-C oxygen picks up a proton from a nearby Lysine amino acid.
The fourth box is Step 3 which is the saturation of the alpha-beta unsaturated double bond between carbons 2 and 3. In this step of the reaction, Lysine deprotonates the oxygen off the alpha carbon and the lone pair of electrons on the oxygen of the alpha carbon comes down reforming the ketone double bond and pushing the lone pair (forming the double bond between the alpha and beta carbon) off, picking up a proton from the nearby Tyrosine amino acid. This reaction results in the formation of alpha-ketoglutarate, NADH+H+/NADPH+H+, and CO2.
Looking at the 3-D structures to the left, two Aspartate amino acids are interacting with two adjacent water molecules (w6 and w8) in the Mn2+ isocitrate porcine IDH complex to deprotonate the alcohol off the alpha-Carbon. The oxidation of the alpha-C also takes place in this picture where NAD+ accepts a hydride resulting in oxalosuccinate. Along with the sp3 to sp2 stereochemical change around the alpha-C, there is a ketone group that is formed form the alcohol group. The formation of this ketone double bond allows for resonance to take place as electrons coming down from the leaving carboxylate group move towards the ketone.
The decarboxylation of oxalosuccinate is a key step in the formation of alpha-ketoglutarate. In this reaction, the lone pair on the adjacent Tyrosine oxygen pulls off the proton of the carboxyl group. This carboxyl group is also referred to as the beta subunit of isocitrate. The deprotonation of the carboxyl proton causes the lone pair of electrons to move down making carbon dioxide and separating from oxalosuccinate. The electrons continue to move towards the alpha carbon pushing the double bond electrons (making the ketone) up to pull a proton off an adjacent Lysine residue. An alpha-beta unsaturated double bond results between carbon 2 and three. As you can see in the picture, the green ion represents either Mg2+ or Mn2+, which is a cofactor necessary for this reaction to occur. The metal-ion forms a little complex through ionic interactions with the oxygen atoms on the fourth and fifth carbons (also known as the gamma subunit of isocitrate).
After carbon dioxide leaves oxalosuccinate in the decarboxylation step, the alcohol on carbon 2 will want to reform the ketone double bond because that’s a more stable form than the enol form which it is in. The reformation of the ketone double bond is started by the deprotonation of that oxygen off the alpha carbon (C#2) by the same Lysine that protonated the oxygen in the first place. The lone pair of electrons moves down kicking off the lone pairs that were making the double bond. This lone pair of electrons pulls a proton off the Tyrosine that deprotonated the carboxyl group in the decarboxylation step. The reason that we can say that the Lys and Tyr residues will be the same from the previous step because they are helping in holding the isocitrate molecule in the active site of the enzyme. These two residues will be able to hydrogen bond back and forth as long as they’re close enough to the substrate.
Left: The isocitrate dehydrogenase enzyme as stated above produces alpha-ketoglutarate, carbon dioxide, and NADH+H+/NADPH+H+. There are three changes that occurred throughout the reaction. The oxidation of Carbon 2, the decarboxylation (loss of carbon dioxide) off Carbon 3, and the formation of a ketone group with a stereochemical change from sp3 to sp2.
Right: Surface view of the active site pocket where isocitrate is bounded by polar amino acids.
Above Left: The Porcine Heart Mitochondrial Enzyme. In this picture you can see that the enzyme is a homeodimer where it is made up of two identical subunits and therefore two identical active sites. Above Right: This is the active site bared down to just isocitrate, Mn2+ and the surrounding interactive amino acids.
(1)Isocitrate binds within the active site to a conserved sequence of about eight amino acids through hydrogen bonds. These acids include (may vary in residue but with similar properties) Tryrosine, Serine, Asparagine, Arginine, Arginine, Arginine, Tyrosine, and Lysine. Their positions on the backbone vary but they are all within a close range (i.e. Arg131 DpIDH and Arg133 PcIDH, Tyr138 DpIDH and Tyr140 PcIDH).
(2)The metal ion (Mg2+,Mn2+) binds to three conserved amino acids through hydrogen bonds. These amino acids include three Aspartate residues.
(3)NAD+ and NADP+ bind within the active site within four regions with similar properties amongst IDH enzymes. These regions vary but are around [250-260], [280-290], [300-330], and [365-380]. Again regions vary but the proximity of regions are conserved.
Above: In this picture, residues Arg110, Arg133, and Arg101 are the three main stabilizing amino acids. They help to hold isocitrate in the active site and in the right orientation for isocitrate dehydrogenase to take place.
Left: Porcine Heart Mitochondrial NADP+-dependent enzyme showing an active spot in green. Porcine enzyme is a homeodimer and has another active site on the other side. Right: Escherichia coli was the first isocitrate dehydrogenase structure derived. There are three active sites. Three isocitrates, one isocitrate in the binding site for NADP+.
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