NaF抑制精氨酸酶催化反应的热动力学研究

Thermokinetic Study on the Inhibition Against Arginase Catalyzed Reaction by NaF

在37℃,pH=7.4～9.4,40mmol?L-1的巴比妥钠-HCl缓冲体系中,利用热动力学方法研究了NaF对精氨酸酶催化L-精氨酸水解反应的抑制作用.实验结果表明,NaF对精氨酸酶反应的抑制作用,属于非竞争性可逆抑制,其抑制率依赖于反应体系的pH值,底物L-精氨酸和外源Mn2+离子对相对抑制率和抑制常数的影响不显著.在pH值为7.4,外源锰离子浓度分别为0和0.167mmol?L-1时的抑制常数分别为1.48和1.84mmol?L-1.F-离子对精氨酸酶的抑制不是与底物L-精氨酸竞争酶的活性位,而是影响了水分子与双核锰簇的桥式配位作用,使反应过程中,作为亲核试剂进攻L-精氨酸胍基碳的羟基离子难于生成或使其浓度减小,从而降低了酶反应活性.

The inhibition against arginase by NaF was studied by thermokinetic method at 37degreesC, pH = 7.4similar to9.4, 40 mmol(.)L(-1) sodium barbiturate-HCl buffer solution. As a result, this experiment indicate that the inhibitory effect of NaF towards arginase is a reversible non-competitive inhibition. For the same reaction, the inhibition rate depends only on pH value of solution, while substrate L-arginine and exogenous Mn2+ ion have no remarkable influence on the inhibition rate. The inhibition constants are 1.48 and 1.84 mmol(.)L(-1) under the condition of exogenous Mn2+ ion concentration of 0 and 0.167 mmol(.)L(-1) respectively. It was suggested that the inhibitory effect do not result from the contest of fluorine anion and L-arginine for the active site of enzyme, and it could be due to that the fluorine anion was in place of mu-OH2 binding as a bridging ligand of the arginase double Mn(II) cluster. This effect obstructs the generation of hydroxyl anion that nucleophilically attacks the guanidino carbon of L-arginine and thus inhibits the reaction activity of arginase.