Molecular modeling of cytochrome P450 2A4 and N-sulfotransferase enzymes in solution.

摘要

Full solution models of two enzymes, generated by molecular dynamics simulations on multiple-nanosecond time scales in solution, are presented. The solution equilibrated models, both in substrate-free and substrate bound forms, are analyzed to address the involvement of active site residues in reaction catalysis.; The solution structure of mouse cytochrome P450 2A4 (CYP2A4), a monooxygenase of deoxysteroids, was obtained using homology modeling and molecular dynamics. The solvent equilibrated CYP2A4 preserves the essential features of CYP450s. A comparison of the model CYP2A4 and CYP2A4 with testosterone bound (CYP2A4/T) illustrate the changes induced by the binding of the substrate. Experimental evidence links four amino acid residues to the catalytic activity, substrate specificity and regioselectivity of this enzyme. Three of the four amino acids are found within contact distance of the testosterone substrate, and therefore may control the binding of the substrate through direct interaction. The location of a water molecule near a bulge in the central helix containing a conserved Thr supports the hypothesis of a proton transfer network.; Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of heparan sulfate, a key step in its biosynthesis. The x-ray crystal structure of the sulfotransferase domain of NDST (NST-1) with PAP served as a basis for a solution model with cofactor PAPS and a model heparan sulfate ligand bound. The solution dynamics of the complex were investigated in the course of a 2 ns simulation. The results confirm the importance of residues Glu642, Lys614, and Lys833 with possible involvement of Thr617 and Thr618, in binding PAPS. Additionally, Lys676 is found in close proximity to the reaction site in the solvated structure. This study illustrates for the first time the possible involvement of water in the NST-1 catalyzed sulfuryl transfer. Three water molecules were found in the active site, coordinated to PAPS, heparan sulfate and the catalytic residues. The solvent equilibrated configuration of the active site was used to explore the mechanism of sulfuryl transfer with a combined quantum mechanical/molecular mechanical (QM/MM) method.