The MD simulations were performed with all the hydrogen bond distances constrained at the beginning, which were then slowly relaxed. orientation. The potential binding models were ultimately identified through the overall evaluation of the docking score of Autodock, MM/PBSA calculations, reaction orientations, and conformational energy penalties (See details in Table S1 and Table S2). Additionally, to avoid the shortcomings of the Autodock program and the conformational analysis method, we tested other docking programs (Gold) with different starting structures and obtained very similar results (see details in Physique S2). Ultimately, conformers M14 and M15 were selected as potential binding models for further analysis. Particularly, M14 was comparable with the binding model proposed by Koch et al. . Since the docking algorithm did not fully account for the structural flexibility of the protein, we performed MD simulations for M14 and M15, using PPO from mitochondria (continuous fluorometric method and compared the results with wild-type position of protogen and the N5 Fiacitabine atom of the FAD. The binding free Fiacitabine energy corresponding to protogen and proto of the transformation process are also labeled (units of kcal/mol). Along the chosen RC, no energy barrier was identified from the free energy profile of the two egress processes. For the substrate protogen, the minimum of the free energy curve was stabilized Fiacitabine with RC?=?3.2 ?, corresponding to the event when the carboxyl oxygen atoms of protogen formed three hydrogen bonds with R98 in tobacco a continuous fluorescence method and were examined in conjunction with the data from the auto-oxidation of protogen in order to examine the occurrence of feedback inhibition. The initial phase of product formation curves was linear, but decreased with time, approaching straight lines (steady says) ( Physique 5A ). However, the product formation was not complete (see the velocities in Physique 5C ). This kind of kinetic time-course exhibited that this enzymatic activity decreased gradually along with the product formation and finally the enzyme became inhibited. The curvilinear functions displayed by the curves were consistent with the presence of a slow, tight-binding inhibitor . This type of kinetic behavior is usually due to a process characterized by the rapid formation of reactant-enzyme complex, followed by a slower dissociation of the product-enzyme complex . The steady states of the product formation curves exhibited a trend of slow rise after the inflection point ( Physique 5A ), which showed that this enzyme was slowly becoming inhibited by the accumulation of product. Open in a separate window Physique 5 A comparison of the conversion of protoporphyrinogen IX to protoporphyrin IX as monitored by fluorescence assay as catalyzed by PPO. A, The enzyme kinetic time-courses with increasing protogen concentrations. The auto-oxidation time-course was excluded from the curve. Reactions were initiated by the addition of enzyme. Data were obtained in the presence of the indicated concentrations of protogen. B, Kinetics of the enzymatic catalysis of a fixed amount of protogen (0.34 PPO (electronic structure calculation with Gaussian03 program at the HF/6-31+G* level . The optimized geometries were used to construct the entire structures of protogen and the final structures of different conformations were optimized with the macrocycle fixed by using conjugated gradient in SYBYL 7.0. The different conformations were used as the starting structures for docking studies. Docking calculations were performed on these conformations with AutoDock4.0 . The protein and ligand structures were prepared with AutoDock Tools . The atomic Gasteiger-Huckel charges were assigned to the ligand and receptor. A total of 256 runs were launched. Most of the parameters for the docking calculation were set to the default values recommended by the software. Each docked structure was scored by the built-in scoring function and was clustered by 0.8 ? of RMSD criterions. For each Rabbit Polyclonal to FANCD2 binding model, molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) was performed (see details in Table S1). Before the MM/PBSA calculation, the complex structure was further refined with the steepest descent algorithm first and then the conjugated gradient algorithm by using the AMBER9 package . During the energy minimization process, the receptor was first fixed and only the ligand was kept free; then the ligand and residue sidechains were kept free; finally all atoms of the system were kept free and refined to a convergence of 0.01 kcal/(mol?). To avoid the drawbacks of the Autodock.