Phosphopentose epimerase (also known as ribulose-phosphate 3-epimerase and ribulose 5-phosphate 3-epimerase, EC 5.1.3.1) encoded in humans by the RPE gene is a metalloprotein that catalyzes the interconversion between D-ribulose 5-phosphate and D-xylulose 5-phosphate.
D-ribulose 5-phosphate ⇌ {\displaystyle \rightleftharpoons } D-xylulose 5-phosphate This reversible conversion is required for carbon fixation in plants – through the Calvin cycle – and for the nonoxidative phase of the pentose phosphate pathway. This enzyme has also been implicated in additional pentose and glucuronate interconversions. In Cupriavidus metallidurans two copies of the gene coding for PPE are known, one is chromosomally encoded P40117, the other one is on a plasmid Q04539. PPE has been found in a wide range of bacteria, archaebacteria, fungi and plants. All the proteins have from 209 to 241 amino acid residues. The enzyme has a TIM barrel structure.
Nomenclature The systematic name of this enzyme class is D-ribulose-5-phosphate 3-epimerase. Other names in common use include
This enzyme participates in 3 metabolic pathways: pentose phosphate pathway, pentose and glucuronate interconversions, and carbon fixation. The human protein containing this domain is the RPE (gene).
Family Phosphopentose epimerase belongs to two protein families of increasing hierarchy. This enzyme belongs to the isomerase family, specifically those racemases and epimerases which act on carbohydrates and their derivatives. In addition, the Structural Classification of Proteins database has defined the “ribulose phosphate binding” superfamily for which this epimerase is a member. Other proteins included in this superfamily are 5‘-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC).
Structure As of late 2007, 4 structures have been solved for this class of enzymes, with PDB accession codes PDB: 1H1Y, PDB: 1H1Z, PDB: 1RPX, and PDB: 1TQJ.
Overall Crystallographic studies have helped elucidate the apoenzyme structure of phosphopentose epimerase. Results of these studies have shown that this enzyme exists as a homodimer in solution. Furthermore, Phosphopentose epimerase folds into a (β/α)8 triosephosphate isomerase (TIM) barrel that includes loops. The core barrel is composed of 8 parallel strands that make up the central beta sheet, with helices located in between consecutive strands. The loops in this structure have been known to regulate substrate specificities. Specifically, the loop that connects helix α6 with strand β6 caps the active site upon binding of the substrate. As previously mentioned, Phosphopentose epimerase is a metalloenzyme. It requires a cofactor for functionality and binds one divalent metal cation per subunit. This enzyme has been shown to use Zn2+ predominantly for catalysis, along with Co2+ and Mn2+. However, human phosphopentose epimerase – which is encoded by the RPE gene - differs in that it binds Fe2+ predominantly in catalysis. Fe2+ is octahedrally coordinated and stabilizes the 2,3-enediolate reaction intermediate observed in the figure.
Active site The β6/α6 loop region interacts with the substrate and regulates access to the active site. Phe147, Gly148, and Ala149 of this region cap the active site once binding has occurred. In addition, the Fe2+ ion is coordinated to His35, His70, Asp37, Asp175, and oxygens O2 and O3 of the substrate. The binding of substrate atoms to the iron cation helps stabilize the complex during catalysis. Mutagenesis studies have also indicated that two aspartic acids are located within the active site and help mediate catalysis through a 1,1-proton transfer reaction. The aspartic acids are the acid/base catalysts. Lastly, once the ligand is attached to the active site, a series of methionines (Met39, Met72, and Met141) restrict further movement through constriction.
Mechanism
Phosphopentose utilizes an acid/base type of catalytic mechanism. The reaction proceeds in such a way that trans-2,3-enediol phosphate is the intermediate. The two aspartic acids mentioned above act as proton donors and acceptors. Asp37 and Asp175 are both hydrogen bonded to the iron cation in the active site. When Asp37 is deprotonated, it attacks a proton on the third carbon of D-ribulose 5-phosphate, which forms the intermediate. In a concerted step, as Asp37 grabs a proton, the carbonyl bond on the substrate grabs a second proton from Asp175 to form a hydroxyl group. The iron complex helps stabilize any additional charges. It is C3 of D-ribulose 5-phosphate which undergoes this epimerization, forming D-xylulose 5-phosphate. The mechanism is clearly demonstrated in the figure.
Function
Calvin cycle Electron microscopy experiments in plants have shown that phosphopentose epimerase localizes to the thylakoid membrane of chloroplasts. This epimerase participates in the third phase of the Calvin cycle, which involves the regeneration of ribulose 1,5-bisphosphate. RuBP is the acceptor of the carbon dioxide (CO2) in the first step of the pathway, which suggests that phosphopentose epimerase regulates flux through the Calvin cycle. Without the regeneration of ribulose 1,5-bisphosphate, the cycle will be unable to continue. Therefore, xylulose 5-phosphate is reversibly converted into ribulose 5-phosphate by this epimerase. Subsequently, phosphoribulose kinase converts ribulose 5-phosphate into ribulose 1,5-bisphosphate.
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