抗坏血酸对兔肌乳酸脱氢酶抑制作用的动力学研究

The kinetic study of the inhibition of rabbit muscle lactate dehydrogenase by ascorbic acid

目的 探讨抗坏血酸对兔肌乳酸脱氢酶 (LDHm)活性的影响及其动力学机制。方法 采用分光光度法追踪酶反应 ,用Lineweaver Burk作图法分析动力学机制。结果 在pH7.0、2 5℃、1 0 1 .3kPa条件下求出LDHm 的KmNADH为 2 .2 5× 1 0 - 5mol/L、KmPyr为 1 .55× 1 0 - 4mol/L、VmaxNADH为 0 .1 1 4A340 /min、VmaxPyr为 0 .1 0 7A340 /min。在酶反应体系中 ,不同浓度的抗坏血酸对LDHm 均具有可逆抑制作用 ,并存在量效关系。随抗坏血酸浓度增加 ,表观KmNADH、表观VmaxNADH均逐渐减小 ,而表观KmNADH/VmaxNADH不变 ;表观KmPyr逐渐增大 ,而表观VmaxPyr不变。结论 抗坏血酸相对于NADH为LDHm 的反竞争抑制剂 ,相对于丙酮酸为LDHm 的竞争性抑制剂 ;抗坏血酸结合于E

Objective To study the effect of ascorbic acid on the activity of rabbit muscle lactate dehydrogenase (LDH m) and obtain the kinetic mechanism of it.Methods The LDH m catalyzed reaction was monitored spectrophotometrically and a set of Lineweaver Burk plots were used to analyze the reaction.Results KmNADH(2.25×10 -5 mol/L)? Km Pyr (1.55×10 -4 mol/L)?Vmax NADH (0.114 A340/min)and Vmax Pyr (0.107 A340/min)were obtained at pH7.0,25℃,101.3kPa? When changing fixed levels of ascorbic acid were added to the reaction system, ascorbic acid functioned as a reversible inhibitor and had stoichiometric relation to its effect. With the increasing of the concentration of the ascorbic acid , apparent Km NADH and apparent Vmax NADH decreased , but apparent Km NADH /Vmax NADH unchanged;whereas apparent Km Pyr increased and apparent Vmax Pyr unchanged. Conclusion Ascorbic acid is an uncompetitive inhibitor of LDH m with respect to NADH and a competitive inhibitor with respect to pyruvate ;Ascorbic acid can bind to E NADH binary complex and thus decrease the activity of LDH m.