人红细胞内核苷二磷酸激酶的酶学特性及动力学研究

Kinetic and mechanistic characterizations of human erythrocytic nucleoside diphosphate kinase

目的:探讨人红细胞(RBC)内核苷二磷酸激酶(NDPK)的酶学动力学特性。方法:利用NDPK催化反应底物二磷酸脱氧腺苷、三磷酸脱氧鸟苷生成三磷酸脱氧腺苷及二磷酸脱氧鸟苷(dADP+dGTP?dATP+dGDP)的酶促反应,通过改变反应温度、热处理温度、pH条件、底物浓度及抑制剂浓度进行NDPK酶学动力学研究。结果:底物动力学研究中,Linewever-Burk双倒数图为两组平行直线。产物抑制动力学研究中,固定反应底物dGTP浓度并改变dADP浓度时,dATP表现为非竞争性抑制作用,固定反应底物dADP浓度并改变dGTP浓度时,dATP表现为竞争性抑制作用。反应遵循乒乓机制。文中实验条件下,NDPK酶促反应最大速度V_(max)约为110μmol·min^(-1)·g^(-1),K_m^(dADP)=1.26 mmol·L^(-1),K_m^(dGTP)=1.94 mmol·L^(-1)。dATP抑制常数K_i约为0.34~0.67 mmol·L^(-1)。结论:NDPK为酶促反应dADP+dGT dAPT+dGDP的一种高亲和性催化酶。

Objective： To investigate the characteristics of enzyme kinetics of NDPK in human erythrocytes.Methods： The enzymatic reaction of dADP＋dGTP？dATP＋dGDP was catalyzed specifically by NDPK.Enzymatic characterizations were investigated by changing the reaction temperatures,heat-treatment temperatures,pH conditions,substrate concentrations and inhibitors concentrations,respectively,based on the above-mentioned reaction.Results： Two sets of parallel lines were obtained when plotting Linewever-Burk double-reciprocal plots in enzyme-substrate kinetic studies.A typical pattern of noncompetitive inhibition was presented when using dADP as the variable substrate and dGTP as the fixed substrate in the presence of various concentrations of dATP.Meanwhile,competitive inhibition was observed when dGTP was the variable substrate and dADP as the fixed substrate in product inhibition studies.The erythrocytic NDPK followed a ＂ping-pong＂ mechanism was reached.An approximate value of Vmax of 110 μmol·min-1·g-1,and KmdADP of 1.26 mmol·L-1,KmdGTP of 1.94 mmol·L-1 were obtained under the conditions in our experiment.Inhibition constant Ki for dATP was considered in the range of 0.34-0.67 mmol·L-1.Conclusion： NDPK is a high-affinity catalytic enzyme for enzg matic reaction of dADP＋dGTP＋dATP＋dGDP.