Interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates: kinetics, mechanism, and analogy to the acylenzyme mechanism.
Interaction of lipoprotein lipase with p-nitrophenyl N-alkylcarbamates: kinetics, mechanism, and analogy to the acylenzyme mechanism.
复制标题
脂蛋白脂肪酶与对硝基苯基 N-烷基氨基甲酸酯的相互作用：动力学、机制以及与酰基酶机制的类比。
DOI：
10.1021/bi00118a025
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Quinn,DM
中科院分区：
文献类型：
--
作者：
Shin,HC;Quinn,DM
酶-氨基甲酸酯复合体(EC)的形成，快速氨甲酰化(KC)先于缓慢的脱氨基甲酸甲酯。因此，在脂蛋白脂肪酶催化对-硝基苯丁酸酯的短期(30分钟)测定中，活性迅速丧失。对硝基苯基氨基甲酸丁酯的抑制作用遵循饱和动力学，测得Kc=5.4±0.9µm，Kc=(4.9±0.7)X 10™2 S-1。较长的缓蚀剂对硝基苯基JV-氨基甲酸辛酯和对硝基苯基YV-十二烷基氨基甲酸酯未观察到饱和动力学。相反，活性损失与抑制剂浓度的伪一级速率常数的曲线图是向上凹陷的，这与抑制剂与酶上的两个位点结合一致。抑制阶段足够快，可以用对硝基苯基氨基甲酸酯滴定酶的活性部位。另一方面，长期试验(>；5h)显示顺序抑制和活性返回阶段，并从活性返回阶段计算k？通过对三阶段机构的微分方程组进行龙格-库塔积分，精确地模拟了长期活动的时间过程。这些方法已被用来表征酶与氨基甲酸酯类抑制剂相互作用的动力学。由于三阶段机制在形式上等同于脂蛋白脂肪酶作用的酰基酶机制，这里描述的结果提供了关于该酶在催化的酰化和去酰化阶段的脂肪酰基专一性的信息。
EC'+ P-^ L. E+ Q formation of the enzyme-carbamate complex (EC), rapid carbamylation (kc) precedes slow decarbamylation. Therefore, in short-term assays (< 30 min) of lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl butyrate, activity is rapidly lost. The inhibition by p-nitrophenyl A-butylcarbamate follows saturation kinetics, which allows determination of Kc= 5.4±0.9 µ and kc=(4.9±0.7) X 10™ 2 s-1. Saturation kinetics are not observed for the longer inhibitors p-nitrophenyl jV-octylcarbamate and p-nitrophenyl yV-dodecylcarbamate. Rather, plots of the pseudo-first-order rate constant for activity loss versus inhibitor concentration are concave upward, consistent with inhibitor bindingto two sites on the enzyme. The inhibition phase is sufficiently rapid that p-nitrophenyl A-octylcarbamate can be used to titrate enzyme active sites. On the other hand, long-term assays (> 5 h) show sequential inhibition and activity return phases, and from the activity return phase k¿ is calculated. The long-term activity time course is accurately simulated by Runge-Kutta integration of the differential equations for the three-stage mechanism. These approaches havebeen used to characterize the kinetics of interaction of the enzyme with the carbamate inhibitors. Because the three-stage mechanism is equivalent in form to the acylenzyme mechanism for lipoprotein lipase action, the results described herein provide information on the fatty acyl specificity of the enzyme in the acylation and deacylation stages of catalysis.