Isocitrate dehydrogenase
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| Found in Escherichia coli | |
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Isocitrate Dehydrogenase
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| Identifiers | |
| Symbol | IDH1 |
| Entrez | 3417 |
| HUGO | 5382 |
| OMIM | 147700 |
| PDB | 1cw7 |
| RefSeq | NM_005896 |
| UniProt | O75874 |
| Other data | |
| EC number | 1.1.1.42 |
| Locus | Chr. 2 q32-qter |
Isocitrate dehydrogenase (EC 1.1.1.42) and (EC 1.1.1.41), also known as IDH, is an enzyme which participates in the citric acid cycle. It catalyzes the third step of the cycle: the oxidative decarboxylation of isocitrate, producing alpha-ketoglutarate (α-ketoglutarate) and CO2 while converting NAD+ to NADH. This is a two-step process, which involves oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by the decarboxylation of the carboxyl group beta to the ketone, forming alpha-ketoglutarate. Another isoform of the enzyme catalyzes the same reaction, however this reaction is unrelated to the citric acid cycle, is carried out in the cytosol as well as the mitochondrion and peroxisome [1] , and uses NADP+ as a cofactor instead of NAD+.
The CAS number for this type of the enzyme is [9028-48-2].
Contents |
[edit] Energetics:
The overall free energy for this reaction with either isoform is -8.4 kJ/mol. IDH lowers the Km (Michaelis constant) of isocitrate without lowering the Vmax (maximum reaction rate - see Michaelis constant for more details).
[edit] Structure:
The NAD-IDH is composed of 3 subunits, is allosterically regulated, and requires an integrated Mg2+ or Mn2+ ion. The closest homologue which has a known structure is the E. coli NADP-dependent IDH, which only has 2 subunits and a 13% identity and 29% similarity based on the amino acid sequences, making it a far cry from human IDH and not suitable for close comparison. All the known NADP-IDHs are homodimers.
[edit] Regulation:
The IDH step of the citric acid cycle, due to its large negative free energy change, is one of the irreversible reactions in the citric acid cycle and therefore must be carefully regulated to avoid unnecessary depletion of isocitrate (and therefore an accumulation of alpha-ketoglutarate). The reaction is stimulated by the simple mechanisms of substrate availability (isocitrate, NAD+ or NADP+, Mg2+ / Mn2+ ), product inhibition (by NADH (or NADPH outside the citric acid cycle) and alpha-ketoglutarate), and competitive feedback inhibition (by ATP).
[edit] Catalytic Mechanism:
Isocitrate + NADP+ Mg2+(metal ion) --> alpha-ketoglutarate + NADPH+H+ + CO2[2][3][4]
[edit] Steps Broken Down:
Within the citric acid cycle, isocitrate, produced from the isomerization of citrate, undergoes both oxidation and decarboxylation. Using the enzyme Isocitrate Dehydrogenase (IDH), isocitrate is held within its active site by surrounding Arginine, Tyrosine, Asparagine, Serine, Threonine, and Aspartic Acid amino acids. The first box shows the overall isocitrate dehydrogenase reaction. The reactants necessary for this enzyme mechanism to work are isocitrate, NAD+/NADP+, and Mn2+ or Mg2+. The products of the reaction are alpha-ketoglutarate, carbon dioxide, and NADH+H+/NADPH+H+.[5] Water molecules are used to help deprotonate the oxygens (O3) of isocitrate.
The second box is Step 1 which is the oxidation of the alpha-C (C#2).[6][7] Oxidation is the first step that isocitrate goes through. In this process[8], 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[9][10], 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[11][12], 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.[13] This reaction results in the formation of alpha-ketoglutarate, NADH+H+/NADPH+H+, and CO2.
[edit] 3-D structure Mechanism:
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.[13] 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.[13] 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.[14]
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.[15]
Above Left: The Porcine Heart Mitochondrial Enzyme.[15] 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.[15]
[edit] Active Site
The Isocitrate Dehydrogenase (IDH) enzyme structure in Escherichia coli was the first structure to be built and understood.[13] Since then Escherichia coli has been used by most researchers to make comparisons to other isocitrate dehydrogenase enzymes. There is much detailed knowledge about this bacterial enzyme and it has been found that most isocitrate dehydrogenases are similar in structure and therefore function. This similarity of structure and function gives reason to believe that the structures are conserved as well as the amino acids.[4] Therefore, the active sites amongst most prokaryotic isocitrate dehydrogenase enzymes should be conserved as well which is observed throughout many studies done on prokaryotic enzymes. Eukaryotic isocitrate dehydrogenase enzymes on the other hand, have not been fully discovered yet. Each dimer of IDH has two active sites.[13] Each active site binds a NAD+/NADP+ molecule and a metal ion (Mg2+,Mn2+). In general, each active site has a conserved sequence of amino acids for each specific binding site. In Desulfotalea psychrophila (DpIDH)[13] and porcine (PcIDH)[15] there are three substrates binded to the active site.
(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).[13]
(2)The metal ion (Mg2+,Mn2+) binds to three conserved amino acids through hydrogen bonds. These amino acids include three Aspartate residues.[13]
(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.[13]
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.[15]
[edit] Monomeric and Dimeric IDH:
When comparing monomer dehydrogenases with dimer dehydrogenases, it's important to note that most enzymes are dimers, specifically homodimers (two identical monomer subunits forming one dimeric unit). In a study done comparing Corynebacterium glutamicum and Escherichia coli[14], monomer and dimer respectively, both enzymes were found to "efficiently catalyze identical reactions." However, C. glutamicum was recorded as having ten times as much activity than E. coli and seven times more affinitive/specific for NADP. C. glutamicum favored NADP+ over NAD+. In terms of stability with response to temperature, both enzymes had a similar Tm or melting temperature at about 55oC to 60oC. However, the monomer C. glutamicum showed a more consistent stability at higher temperatures, which was expected. The dimer E. coli showed stability at a higher temperature than normal due to the interactions between the two monomeric subunits.
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.[15] 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+.
[edit] See also
[edit] References
</references>
- ^ Corpas FJ et al (1999). "Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence". Plant Physiology 121 (3): 921-928.. doi:.
- ^ McMurry, John E., and Tadhg P. Begley. The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company, 2005. 189-190.
- ^ Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and Company, 2005. 609-611.
- ^ a b Yasutake Y, Watanabe S, Yao M, Takada Y, Fukunaga N, Tanaka I (2003). "Crystal Structure of the Monomeric Isocitrate Dehydrogenase in the Presence of NADP+". Journal of Biological Chemistry 278 (38): 36897-36904. doi:.
- ^ Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and Company, 2005. 609-611.
- ^ McMurry, John E.; Tadhg P. Begley (2005). The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company, 189-190.
- ^ Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and Company, 2005. 609-611.
- ^ McMurry, John E., and Tadhg P. Begley. The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company, 2005. 189-190.
- ^ McMurry, John E., and Tadhg P. Begley. The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company, 2005. 189-190.
- ^ Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and Company, 2005. 609-611.
- ^ McMurry, John E., and Tadhg P. Begley. The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company, 2005. 189-190.
- ^ Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and Company, 2005. 609-611.
- ^ a b c d e f g h i Fedoy AE, Yang N, Martinez HKSL, Steen IH (2007). "Structural and Functional Properties of Isocitrate Dehydrogenase from the Psychrophilic Bacterium Desulfotalea psychrophila Reveal a Cold-active Enzyme with an Unusual High Thermal Stability". Journal of Molecular Biology 372 (372): 130-149. doi:.
- ^ a b Chen R, Yang H (2000). "A Highly Specific Monomeric Isocitrate Dehydrogenase from Corynebacterium glutamicum". Archives of Biochemistry and Biophysics 383 (2): 238-245. doi:.
- ^ a b c d e f g h i j k Ceccarelli C, Neil B (2002). "The Crystal Structure of Porcine Mitochondrial NADP+-dependent Isocitrate Dehydrogenase Complexed with Mn2+ and Isocitrate". Journal of Biological Chemistry 277 (45): 43454-43462. doi:.
- IUBMB entry for 1.1.1.42
- BRENDA references for 1.1.1.42 (Recommended.)
- PubMed references for 1.1.1.42
- PubMed Central references for 1.1.1.42
- Google Scholar references for 1.1.1.42
[edit] External links
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