氨基酰化酶中金属锌离子的功能作用

 氨基酰化酶是含锌金属酶。该酶每摩尔蛋白中含2摩尔Zn(Ⅱ)离子。金属鳌合剂与酶作用,通过竞争螯合Zn(Ⅱ)离子使酶活力下降。残余活力与残留金属含量呈正相关。竞争螯合的结果,生成不含金属的脱辅基酶蛋白,并导致酶活力的丧失。脱辅基酶由于加入Zn(Ⅱ)离子而恢复其活力。实验表明金属锌离子是氨基酰化酶催化活力所必需。与Zn(Ⅱ)离子相似,Co(Ⅱ)离子也可与脱辅基酶相结合并使之复活。 在190—240nm区域内对比了天然酶、脱辅基酶蛋白与Co(Ⅱ)置换氨基酰化酶的圆二色谱。远紫外圆二色谱表明,与天然酶相比,在脱辅基酶中由于金属离子的丧失导致主链构象发生变化,其中α螺旋增加约7％。因而锌离子(钴离子)对蛋白主链的反应最适构象有一定的稳定作用。脱辅基酶与Co(Ⅱ)离子结合,酶的主链构象恢复至与天然酶几近相同。可认为这是促使酶复活的内在因素。

THE FUNCTIONAL ROLE OF ZINC IN AMINOACYLASE

Zinc plays different roles in metalloenzeme. It may be directly de involved in the catalysis process as a catalytic site. It may also regulate catalytic activity by affecting the active site conformation.Aminoacylase from pig kidney is a metalloenzyme which contains 2 moles of zinc per mole of protein.The removal of Zn(Ⅱ)was performed by dialysis of the enzyme (1.2mg/ml) in 0.1 mol/L phosphate buffer, pH 7.3, against lmol/L 1 ,10-phenanthroline at 24℃ Affer appropriate time intervals,the zinc contens and the activity were determined. The results demonstrate that the loss of activity was exactly proportional to the amount of zinc removed by dialysis.Reactivation of Zn-free inactive aminoacylase was performed by incubation for 20 minutes at 37℃ in the presence of 1×10-4mol/L Zn(Ⅱ) or Co(Ⅱ) ions.Circular dichroism spectra (CD) were used to study the effect of zinc on the structural stability of protein.The CD spectra were measured with a JASCO J-500 C spectropolarimeter at 18℃ in the range between 190 and 240nm, and the fractions of α-helix(fα),β-pleated sheet(fβ)and random coil (fR) in protein were computed with the Y.H.Chen's method.Comparison of the CD spectra of the native aminoacylase and the Zn-free apoenzyme showed that, upon removal of the Zn(Ⅱ) from the active site considerable changes in conformation, including about 7% of increase in a-helix, were induced.These results suggested that the zinc may contribute to the structural stability of the protein. It also worth noting that the conformation of the Co(Ⅱ)-substituted enzyme is similar to the native enzyme, and this may be the reason for restoring of the enzymatic activity of metal-free apoenzyme upon the addition of Co(Ⅱ) ion.