Phosphopentose epimerase

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.[5][6] Furthermore, Phosphopentose epimerase folds into a (β/α)₈ triosephosphate isomerase (TIM) barrel that includes loops.[2] 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.[2]

As previously mentioned, Phosphopentose epimerase is a metalloenzyme. It requires a cofactor for functionality and binds one divalent metal cation per subunit.[7] This enzyme has been shown to use Zn²⁺ predominantly for catalysis, along with Co²⁺ and Mn²⁺.[2] 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.[2]

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.[2] 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.[1] 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.[8]

Electron microscopy experiments in plants have shown that phosphopentose epimerase localizes to the thylakoid membrane of chloroplasts.[12] 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.[11]

The reactions of the pentose phosphate pathway (PPP) take place in the cytoplasm. Phosphopentose epimerase specifically affects the nonoxidative portion of the pathway, which involves the production of various sugars and precursors.[2] This enzyme converts ribulose 5-phosphate into the appropriate epimer for the transketolase reaction, xylulose 5-phosphate.[11] Therefore, the reaction that occurs in the pentose phosphate pathway is exactly the reverse of the reaction which occurs in the Calvin cycle. The mechanism remains the same and involves the formation of an enediolate intermediate.

Due to its involvement in this pathway, phosphopentose epimerase is an important enzyme for the cellular response to oxidative stress.[2] The generation of NADPH by the pentose phosphate pathway helps protect cells against reactive oxygen species. NADPH is able to reduce glutathione, which detoxifies the body by producing water from hydrogen peroxide (H₂O₂).[2] Therefore, not only does phosphopentose epimerase alter flux through the PPP, but it also prevents buildup of peroxides.