Inhibition Mechanism Of Gallic Acid And Catechinic Phenols On Mucor Miehei Lipase

Lipase(E.C.3.1.1.3),also known as triacylglyceryl hydrolase,which can be found almost universally in every corner of the biological community.As an important metabolic enzyme in life,all of them belong to serine hydrolase.They have a conserved pentapeptide in structure,so that all can catalyze the same reaction even if the sequence is different.Lipase inhibitors are substances which can inhibit lipase activity,so they can control and treat obesity by suppressing dietary fat in the process of decomposition,reducing the body's absorption of fat.This is the main strategy for treating obesity,using nutritional digestion and absorption inhibitors,such as Orlista,which is the only clinically approved treatment for obesity.However,long-term use of Orlista can cause a range of side effects.Therefore,the search for low toxicity and high efficiency lipase inhibitors has become a hot topic.The enzymatic properties of Mucor miehei Lipase(MML)as a catalyst for the hydrolysis of long chain fatty acid acyl Ester 4-Nitrophenyl palmitate(p-PNC16)were studied.The experimental results showed that the optimum reaction temperature was 35 ?,the optimum reaction pH was in the range of 7.8-8.2,and the enzyme activity was high in the range of pH 8.0-9.0 and 30-40?.The kinetic parameters Km and Vm are 0.59 mmol/L and 24.69 mm mol/L/min,respectively.The effect of 16 common metal ions and acid ions at a given concentration(1-10 mmol/L)on the reaction.The results showed that Li +,Na +,K+,SO42-,Cl-,NO3-had little effect on MML activity,while Mg2+,Ca2+,Ba2+ and Mn2+ increased enzyme activity by 64.2%,51.3%,116.2%and 218.7%respectively at ion concentration of 1.0 mol/L.Fe3+,Al3+,Zn2+,Hg2+ and Cu2+ acted as inhibitors of enzyme activity,which decreased by 88.5 percent,82.7 percent,8.9 percent,71.2 percent and 22.9 percent,respectively,at ion concentrations of 1.0 mmol/L.In addition,this study focused on the inhibition of Gallic acid(G),(-)-Epicatechin(EC),(-)-Epigallocatechin(EGC)and(-)-Epigallocatechin gallate(EGCG)on lipase.The four phenolic substances were similar in structure.IC50 for G,EC,EGCand EGCG were 1.29,0.23,0.026,0.017 mmol/L,respectively.G showed Con-competitive inhibition of enzymes with an inhibition constant of 1.29 mmol/L.G,EC,EGCand EGCG showed mixed inhibition with inhibition constants of 0.146 mmol/L and 0.375 mmol/L,0.014 and 0.057 mol/L,0.012 and 0.039 mol/L.Based on the previous research,the dynamics of deactivation and inhibition of four effectors on MML was studied,and the action model was established and the microvelocity constant was measured.Among them,G is non-competitive,indicating that the enzyme,the substrate and G can be reversibly and independently combined into an inactive ternary complex.The deactivation rate constant of catechins was higher than that of enzyme-substrate complex,which showed that the inhibition of enzyme was protective.All four effects can be combined with MML to reduce enzyme activity.Using MOE software to simulate the binding of effector and MML,the binding ability of enzyme protein and small molecular ligands was from large to small,EGCG,EGC,EC and G.All four effectors affect the important amino acid residues of the active centers.Fluorescent quenching also showed consistent results.The binding constant(KA)and binding site(n)of EGCG,which is the strongest inhibition of enzymes were higher than others.In light of the foregoing,On the basis of the research of enzyme properties of mML catalyzed p-PNC16 reaction,this paper establishes a lipozyme inhibitor screening system,which can be applied to the screening of lipase inhibitors and chemosynthesis inhibitors from natural sources.On the other hand,the inhibitory effect of four phenols on MML was studied,and the potential relationship between the inhibitory effect and its structure was discussed.This provides theoretical basis for screening and synthesis of lipase inhibitors.