第208位异亮氨酸对人细胞色素P4502A6尼古丁代谢活性的影响

Effect of isoleusine 208 on nicotine 5'-hydroxylation by human CYP2A6

目的探讨人体尼古丁主要代谢酶细胞色素P450(CYP)2A6及其同族成员CYP2A13肽链结构中,影响其尼古丁5′-羟化代谢活性的关键性氨基酸残基。方法使用前期制备的CYP2A6和CYP2A13系列氨基酸互换突变体:CYP2A6V117A,CYP2A6G164H,CYP2A6I208S,CYP2A6R372H和CYP2A6S465P以及CYP2A13A117V,CYP2A13H164G,CYP2A13S208I,CYP2A13H372R和CYP2A13P465S,比较其与相应野生蛋白酶的尼古丁5′-羟化催化反应的动力学参数。结果各突变体对2个CYP2A蛋白酶的尼古丁代谢活性影响不同。对于CYP2A6,I208S突变对酶活性的影响显著,导致表观反应常数Km及最大反应速度Vmax由野生型62.25μmol.L-1和6.53mol.min-1.mol-1变化为345μmol.L-1和2.19mol.min-1.mol-1,但该位点对CYP2A13酶活性无显著影响;对于CYP2A13,H372R突变对酶活性的影响最为显著,导致Km及Vmax由野生型的26.01μmol.L-1和24.51mol.min-1.mol-1变为148.7μmol.L-1和6.11mol.min-1.mol-1,此位点对CYP2A6无显著影响。其他位点突变对酶活性影响较小或不显著。结论CYP2A家族蛋白中,I208与H372分别是影响CYP2A6和CYP2A13对尼古丁代谢的关键残基。对于同家族蛋白酶而言,关键性氨基酸的作用并不总是一一对应。

AIM To identify potential amino acid residues that contribute to different catalytic characteristics of CYP2A6 and CYP2A13 enzymes in nicotine metabolism.METHODSWild type of CYP2A6 and CYP2A13 and their mutants CYP2A6V117A,CYP2A6G164H,CYP2A6I208S,CYP2A6R372H,CYP2A6S465P and CYP2A13A117V,CYP2A13H164G,CYP2A13S208I,CYP2A13H372A and CYP2A13P465S,were subjected kinetic analysis in nicotine 5＇-hydroxylation.RESULTS For CYP2A6,substitution of isoleucine 208 to serine caused dramatic kinetic property changes with Km and Vmax varied from 62.25 μmol·L-1 and 6.53 mol·min-1·mol-1 to 345 μmol·L-1 and 2.19 mol·min-1·mol-1.However,the corresponding serine 208 to isoleucine mutation did not heavily affect the enzyme activity in CYP2A13.The histidine 372 to arginine mutation resulted in a remarkable catalytic efficiency decrease with Km and Vmax changes from 26.01 μmol·L-1 and 24.51 mol·min-1·mol-1 to 148.7 μmol·L-1 and 6.11 mol·min-1·mol-1 in CYP2A13,but the switching of argenine 372 to histidine did not show expected corresponding crucial influence in CYP2A6 activity.Substitutions on the other positions changed enzyme activities in different rates.CONCLUSION The isoleucine 208 is crucial to human CYP2A6,while the 372 histidine is a key amino acid residue for CYP2A13 in nicotine 5＇-hydroxylation.