Theoretical Studies On The Cytochrome Enzyme P450 4Z1 Catalyzing The Regioselectivity Of Lauric Acid

Objective: Cytochrome P450(Cytochrome P450,CYP)as a very important catalyst occupies an extremely important position in the human metabolic process,it can participate in a variety of metabolic processes including hydroxylation reactions,is the drug pharmacokinetic And the main source of response variability.CYP enzymes in humans are classified according to sequence similarity.Cytochrome P450 4(Cytochrome P450 4,CYP 4)family is the second largest human CYP family,except CYP 4F8,CYP 4F12,CYP 4X1 and CYP 4Z1,most CYP4 enzymes wereω-hydroxylase.CYP 4Z1 is a new member of the CYP4 family.It is one of the "orphan" cytochrome enzymes that is rarely studied at present.Its overexpression can promote the occurrence of breast cancer and is related to the treatment and poor prognosis of certain cancers.Potential drug targets for cancer treatment.Lauric acid plays an important role in the treatment of various cancers and the adjustment of postoperative rehabilitation.According to reports,the in-chain hydroxylation of lauric acid was catalyzed by CYP 4Z1,but its catalytic mechanism is not yet clear.This paper explores the regioselective characteristics of CYP 4Z1 catalyzed lauric acid through theoretical calculations,and provides theoretical guidance for studying the catalytic mechanism of other CYP 4 enzyme families..Methods: First,the QM calculations were used to determined the hydrogen abstraction barriers of the seven potential catalytic sites at the end of lauric acid,the structure of the reactants and the transition states were determined,and the effect of weak interaction was determined by adding a dispersion correction calculation.Use homology modeling to establish a three-dimensional model of human CYP 4Z1,use molecular dynamics methods to optimize the generated model.After determining the optimal model for modeling,molecular docking was performed to determine the best binding conformation of lauric acid and CYP 4Z1.By setting the presence of F313 A mutant to explore the effect of mutation on molecular docking results and determine the optimal binding conformation of lauric acid and CYP 4Z1 in the mutant.Compare the similarities and differences of key amino acids between wild-type and mutant systems,and compare the best binding conformation of the two.Four classicalmolecular dynamics simulation systems containing the F313 A mutation system were set up to investigate the dynamic process of lauric acid catalyzed by CYP 4Z1 through a simulation of up to 400 ns.Using enhanced sampling simulation to study the role of aromatic amino acids in the process of stabilizing the substrate,looking for the best pathway for CYP 4Z1 to catalyze lauric acid.Finally,the ONIOM calculation method was used to compare the free energy of the CYP 4Z1-lauric acid complex system.Results and conclusions: QM calculations based on the heme and lauric acid models show that the potential hydrogenation barriers of the hydroxylation sites range from56.2 to 85.1 k J / mol,with the lowest barrier at the ω-4 site.The results of molecular docking indicate that Ser383,Ser113,and Arg487 near the active site maintain the stability of lauric acid at the active site by forming hydrogen bonds with lauric acid.Molecular dynamics simulation results show that the common interaction formed by the heme plane and the π-π interaction pushes the ω-4 site closer to the active oxygen,which is consistent with the experimental observations.When Phe313 involved in the formation of π-π interaction is mutated to Ala313,the effect on catalytic selectivity disappears.Combined with free energy analysis,it is shown that the aromatic and polar amino acids around the active site of CYP 4Z1 play an important role in catalytic selectivity.The results of secondary structure analysis and principal component analysis based on wild-type and mutant systems show that the presence of Phe313 near the active site during the catalytic process will cause the structure away from the active site to change to adapt to the changes in the energy of the catalytic system.Channel analysis indicates the gate effect of aromatic amino acids.Enhanced sampling simulations explore how Phe313 and Trp120 affect the energy of the complex system,and determine the feasible pathway for CYP 4Z1 to catalyze the omega-4 site of lauric acid.The ONIOM calculation method calculates that the free energy of the composite system at the catalytic ω-4 site is less than the free energy of the catalytic ω site system.