Substrate Channeling of Prostaglandin H₂ on the Stereochemical Control of a Cascade Cyclization Route

Enzymes can carry out effective rate accelerations by virtue of their ability to utilize substrate-channeling forces to act as a mechanochemical valve. Such a channeling process is treated quantitatively using the key aspects of the free energy landscape; the balance between substrate positioning and conformational changes reflects the severe geometric and electronic requirements for the relatively tight transition state. The observed <i>k</i>_cat/<i>K</i>_M of about 10⁶ M^–1 s^–1 for PGH₂ cyclization has been revealed to be brought about by bringing together two properly oriented reactants of substrate and enzyme regarding the magnitude significance of the contribution from outer- and inner-binding stereopopulation along the free-energy channeling pathway and thus shapes the cascade cyclization route, enforcing precise spatial and temporal control. The apparent constant <i>k</i>_cat of many P450 reactions involving the heme catalytic cycle, which is often on the order of 10¹–10² s^–1 and is usually attributed to compound 0 to compound I formation, may be in large part a consequence of channeling conformation changes toward the rate-limiting state that is made possible by preorganizing the proximal hydrogen-bonding pattern of the amide groups to the cysteine sulfur, and to the push–pull modulation of the relevant heme axial ligation and activation

Homology Modeling and Molecular Dynamics Simulation Combined with X‑ray Solution Scattering Defining Protein Structures of Thromboxane and Prostacyclin Synthases

A combination of molecular dynamics (MD) simulations and X-ray scattering (SAXS) has emerged as the approach of choice for studying protein structures and dynamics in solution. This approach has potential applications for membrane proteins that neither are soluble nor form crystals easily. We explore the water-coupled dynamic structures of thromboxane synthase (TXAS) and prostacyclin synthase (PGIS) from scanning HPLC–SAXS measurements combined with MD ensemble analyses. Both proteins are heme-containing enzymes in the cytochrome P450 family, known as prostaglandin H₂ (PGH₂) isomerase, with counter-functions in regulation of platelet aggregation. Currently, the X-ray crystallographic structures of PGIS are available, but those for TXAS are not. The use of homology modeling of the TXAS structure with ns−μs explicit water solvation MD simulations allows much more accurate estimation of the configuration space with loop motion and origin of the protein behaviors in solution. In contrast to the stability of the conserved PGIS structure in solution, the pronounced TXAS flexibility has been revealed to have unstructured loop regions in connection with the characteristic P450 structural elements. The MD-derived and experimental-solution SAXS results are in excellent agreement. The significant protein internal motions, whole-molecule structures, and potential problems with protein folding, crystallization, and functionality are examined

On the Stereoselectivity of Ring-Opening Metathesis Polymerization (ROMP) of <i>N</i>‑Arylpyrrolidine-Fused Cyclobutenes with Molybdenum– and Ruthenium–Alkylidene Catalyst

Ring-opening metathesis polymerization (ROMP) of cyclobutenes fused with <i>N</i>-arylpyrrolidene with Schrock–Hoveyda catalyst containing a racemic biphenolate ligand [Mo­(N-2,6-<i>i</i>-Pr₂C₆H₃)­(CHCMe₂Ph)­(biphenolate)] gives polycyclobutenes with homogeneous tacticity and predominantly double bonds in <i>Z</i>-configuration. Reactions of the same substrates with the first-generation Grubbs catalyst [(Cy₃P)₂Cl₂RuCHPh] or Schrock molybdenum carbene with monodendate alkoxy ligands [Mo­(N-2,6-<i>i</i>-Pr₂C₆H₃)­(CHCMe₂Ph)­(OCMe­(CF₃)₂] yield the corresponding polycyclobutene containing a mixture of <i>Z</i>- and <i>E</i>-double bonds. Upon diimide reduction, all these polycyclobutenes give the same tactic hydrogenated polymers, indicating that the stereochemistry at the asymmetric carbons remains the same in all these reactions. The stereoselectivities of ROMP with cyclobutenes and with norbornenes are compared, and the plausible mechanisms are proposed

Probing Ligand Binding to Thromboxane Synthase

Various fluorescence experiments and computer simulations were utilized to gain further understanding of thromboxane A₂ synthase (TXAS), which catalyzes an isomerization of prostaglandins H₂ to give rise to thromboxane A₂ along with a fragmentation reaction to 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde. In this study, 2-<i>p</i>-toluidinylnaphthalene-6-sulfonic acid (TNS) was utilized as a probe to assess the spatial relationship and binding dynamics of ligand–TXAS interactions by steady-state and time-resolved fluorescence spectroscopy. The proximity between TNS and each of the five tryptophan (Trp) residues in TXAS was examined through the fluorescence quenching of Trp by TNS via an energy transfer process. The fluorescence quenching of Trp by TNS was abolished in the W65F mutant, indicating that Trp65 is the major contributor to account for energy transfer with TNS. Furthermore, both competitive binding experiments and the computer-simulated TXAS structure with clotrimazole as a heme ligand strongly suggest that TXAS has a large active site that can simultaneously accommodate TNS and clotrimazole without mutual interaction between TNS and heme. Displacement of TNS by Nile Red, a fluorescence dye sensitive to environmental polarity, indicates that the TNS binding site in TXAS is likely to be hydrophobic. The Phe cluster packing near the binding site of TNS may be involved in facilitating the binding of multiple ligands to the large active site of TXAS

Probing Water Environment of Trp59 in Ribonuclease T1: Insight of the Structure–Water Network Relationship

In this study, we used the tryptophan analogue, (2,7-aza)­Trp, which exhibits water catalyzed proton transfer isomerization among N(1)-H, N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59 (Trp59) near the connecting loop region of ribonuclease Tl (RNase T1) by replacing the tryptophan with (2,7-aza)­Trp. The resulting (2,7-aza)­Trp59 triple emission bands and their associated relaxation dynamics, together with relevant data of 7-azatryptophan and molecular dynamics (MD) simulation, lead us to propose two Trp59 containing conformers in RNase T1, namely, the loop-close and loop-open forms. Water is rich in the loop-open form around the proximity of (2,7-aza)­Trp59, which catalyzes (2,7-aza)­Trp59 proton transfer in the excited state, giving both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer in the loop-open form, supported by the MD simulation, is mainly due to the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. The loop-close form is relatively tight in space, which squeezes water molecules out of the interface of α-helix and β2 strand, joined by the connecting loop region; accordingly, the water-scant environment leads to the sole existence of the N(1)-H isomer emission. MD simulation also points out that the Trp-water pairs appear to preferentially participate in a hydrogen bond network incorporating polar amino acid moieties on the protein surface and bulk waters, providing the structural dynamic features of the connecting loop region in RNase T1