MD-QM Simulation on the Prostaglandin H2 Isomerization by Prostacyclin Synthase Supports the Water Reshuffling Proton Coupled Electron Transfer Mechanism

• Main Authors: Weng, Shih-Hui, 翁詩惠
• Other Authors: Yang, Hsiao-Ching
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/22952887962922598443

碩士 === 輔仁大學 === 化學系 === 102 === Water is a remarkable substance to sustain life as well as a latent energy autocatalyst. Its associated properties still remain elusive, specifically in the micro-solvated water structures within the biological systems. This study reports the water reshuffling proton-coupled electron transport (PCET) mechanism in prostacyclin synthase (PGIS) whereby its function converts prostaglandin H2 to prostacyclin (PGI2), a potent vasodilator and anti-platelet clotting mediator in cardiovascular homeostasis. PGIS is a cytochrome P450 enzyme, but differs from canonical P450s in which the well-conserved Thr/Ser residue for the distal heme pocket, where the substrate binds is shaped by a Trp residue whose indole ring parallels to the porphyrin, as revealed by the crystallography structures. On the basis of our previous investigations, it’s worthy to note that PGIS exhibits a ligand-specific heme conformational change to accommodate the substrate binding, in particular of the water rearrangement in the heme site. We had proved this water-network structure change in heme site by resonance Raman (RR) spectra. Herein, we focus our attention on a conserve water bridge structure connecting between the Asn and Trp residues in the distal heme pocket of hPGIS upon PGH2 binding by means of Quantum Mechanical/ Molecular Dynamics (MD) simulations. The nitrogen atom of Trp residue forms hydrogen network with two waters, which in turn form H-bonds to N287 with subsequent water molecule connection to the substrate head endoperoxide. Theoretical calculations suggest this water bridge structure as a PCET shuttle delivering proton/electron to confer high product fidelity for PGIS catalysis. Mimicking this PCET shuttling was done by mutagenesis studies, replacing Asn to Ala, Ser or His suppressed the activity of PGIS with 70-80% loss in kcat and 50-60% reduction in KM, whereas mutation of Trp to Gly, Ala, or Phe results in ~90% loss in kcat accompanied by fold increase in KM, rendering Trp mutants very inefficient enzymes. These results, together with subtle changes in virtual site-directed mutagenesis demonstrate the crucial role of the Trp residue whose indole ring is rather susceptible to ensure hydrogen-bonding formation to facilitate PCET shuttling for optimal catalysis. As manifested, probing the water environment of a specific site in protein should thus pave a way to shed light on the underlying mechanism.