PAR对氨基酰化酶不可逆抑制动力学研究

本文将邹氏的在酶的活性修饰剂存在下的底物反应动力学理论应用于氨基酰化酶被金属螯合剂PAR脱锌而失活的动力学研究。通过对不同浓度的PAR存在下底物反应过程和含有PAR的不同浓度的底物中酶促反应的分析,讨论了PAR对氨基酰化酶的脱锌机制。这一过程很可能按如下机制进行:首先,PAR与酶分子活性部位的锌结合,形成一复合物,这一步是较快的反应,然后发生一个可逆的构象变化,最后是不可逆的去锌步骤。锌的存在显然稳定了酶活性部位的构象,而这正是酶活性所必需的。     

胍和脲溶液对氨基酰化酶活性和构象的影响

 本文研究了不同浓度盐酸胍和脲溶液对猪肾氨基酰化酶活性和构象的影响。研究结果表明,在低浓度的胍和脲溶液中(小于2mol/L),酶分子的整体构象变化的程度与活力变化的程度基本是平行的;而在高浓度的胍和脲溶液中(2mol/L以上),失活程度稍大于构象变化的程度。这些结果与分子量和亚基组成基本相同,但不含金属配基的肌酸激酶的结果,以及小分子量的胰凝乳蛋白酶和牛胰核糖核酸酶的结果相比较来看,可以认为配基锌离子的存在对酶分子的活性部位区域构象的稳定作用有一定的贡献,致使氨基酰化酶的活性部位的构象状态不象后三种酶那样脆弱。同时,我们还发现锌离子的存在对酶分子整体构象的稳定性上贡献很小。     

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。     

氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去圬剂十二烷基硫酸锂（ＬＤＳ）溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液（０．６ｍｍｏｌ／Ｌ）中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化。而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度,在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。从上述研究结果,可以认为含锌氨基酰化酶的活性部位也具有相对的柔性。     

溴化+烷基三甲基铵滴定时氨基酰化酶的失活与构象变化的比较

研究了阳离子去污剂-溴化+烷基三甲基铵变性时氨基酰酶的失活与构象变化.当用溴化+烷基三甲基铵滴定氨基酰化酶时,随着去污剂浓度增大,酶的活力逐渐丧失,至50mmolL时酶完全失活.用荧光发射光谱(295nm激发)的方法监测了氨基酰化酶的构象变化.发现氨基酰化酶失活先于构象变化.从这一结果看来.金属酶的活性部位构象可能也是比整个分子的构象具有较大的柔性或运动性.     

溴化+烷基三甲基铵滴定时氨基酰化酶的失活与构象变化的比较

研究了阳离子去污剂-溴化+烷基三甲基铵变性时氨基酰酶的失活与构象变化.当用溴化+烷基三甲基铵滴定氨基酰化酶时,随着去污剂浓度增大,酶的活力逐渐丧失,至50mmolL时酶完全失活.用荧光发射光谱(295nm激发)的方法监测了氨基酰化酶的构象变化.发现氨基酰化酶失活先于构象变化.从这一结果看来.金属酶的活性部位构象可能也是比整个分子的构象具有较大的柔性或运动性.     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ（ｈｏｌｏ－ＳＯＤ）和脱铜锌ＳＯＤ（ａｐｏ－ＳＯＤ）在不同浓度胍溶液中的去折叠及活力变化．结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失．提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏．     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ和脱铜锌ＳＯＤ在不同浓度胍溶液中的去折叠及活力变化。结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失。提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏     

大肠杆菌精氨酰—tRNA合成酶变种ArgRS381KA的基本性质

本文研究了Ｌｙ３８１变为Ａｌａ的精氨酰－ｔＲＮＡ合成酶（ＡｒｇＲＳ）变种ＡｒｇＲＳ３８１ＫＡ的最适ｐＨ和稳态动力学性质;比较了此酶与天然酶ＡｒｇＲＳ的荧光光谱性质和热稳定性。实验结果表明ＡｒｇＲＳ３８１ＫＡ的氨酰化活力和ＡＴＰ￣ＰＰｉ交换活力的最适ｐＨ分别为８．０和７．０,与天然酶相同;ＡｒｇＲＳ３８１ＫＡ的氨酰化活力对精氨酸、ＡＴＰ和ｔＲＮＡ＾Ａｒｇ的Ｋｍ分别为１２μｍｏｌ／Ｌ、０．３ｍｍｏｌ／     

氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去污剂十二烷基硫酸溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化,而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度。在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions

The equilibrium unfolding of pig kidney aminoacylase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmCl, less than 1.0 M, the fluorescence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concentrations of denaturant (between 1.2 and 1.6 M GdmCl). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Further changes in the fluorescence properties occurred at higher concentrations of GdmCl, more than 1.6 M, with a decrease in emission intensity and a significant red shift of the emission maximum from 340 to 354 nm. In this stage, the secondary structure was completely broken. A study of apo-enzyme (Zn2+-free enzyme) produced similar results. However, comparison of the changes of the fluorescence emission spectra of native (Holo-) enzyme with Zn2+-free (Apo-) enzyme at low GdmCl concentrations showed that the structure of the Holo-enzyme was more stable than that of the Apo-enzyme.     

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.     

Trichloroacetic acid-induced molten globule state of aminoacylase from pig kidney

The trichloroacetic acid (TCA)-induced unfolding of aminoacylase was investigated by measurement of aggregation, enzyme activity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate (ANS) binding, circular dichroism, and native polyacrylamide gel electrophoresis. The results showed that TCA caused inactivation and unfolding of aminoacylase. Intrinsic fluorescence results demonstrated that the TCA-induced transition of aminoacylase was characterized by two distinct stages during which the fluorescence emission maxima first redshifted to 338 nm and then blueshifted to 332 nm, close to that of native protein. ANS binding measurements revealed that TCA-denatured aminoacylase had a large hydrophobic area for TCA concentration near 2 mM. Comparison of the relative changes in wavelength shift and in the ANS intensity suggested the formation of a stable molten globule state of aminoacylase with a slightly disrupted tertiary structure and more hydrophobic surface than the native protein. Far-UV circular dichroism results provided further support that TCA induced the formation of two partially folded intermediates each with an enhanced native-like secondary structure. The results collectively suggest that a TCA-induced molten globule state is formed and stabilized during unfolding of aminoacylase and that association of the molten globule state may account for precipitation of the protein when denatured by TCA.     

Aspartate-induced aminoacylase folding and forming of molten globule

Aspartate is an osmolyte found in some marine invertebrates and cyclostome fish. The aspartate-induced unfolding of N-acylamino acid amido hydrolase (aminoacylase) has been studied by measuring enzyme activity, fluorescence emission spectra, 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that aspartate caused the inactivation and unfolding of aminoacylase. Surprisingly, increasing concentration of aspartate showed the "acid-induced folding", which used to be seen only in strong acids or salts at much lower pH. Although aspartate has the pI of 2.77 that is the lowest among all the free amino acids, it is actually a weak acid. It is thus of great interest why it causes this phenomenon to happen. The relative change of intrinsic fluorescence and ANS binding spectra have shown that there existed a stable molten globule state of aminoacylase with slightly disrupted tertiary structure and more hydrophobic surface. The molten globule state indicates that intermediates existed during aminoacylase refolding process. Unlike the other acids, such as trichloroacetic acid, there is no precipitation observed as the aspartate concentrations increased. It suggests the aspartate anions have an osmotic effect for the molten globule formed during unfolding process. Binding of aspartate anion to the protonated protein, which minimizes the intramolecular repulsion, might explain the osmotic effect of this amino acid in the nature. The results also showed the Apo-aminoacylase followed similar rules as Holo-enzyme, which suggested the zinc ion may play more important roles on activity other than structure.     

Role of osmolytes as chemical chaperones during the refolding of aminoacylase.

The refolding and reactivation of aminoacylase is particularly difficult because of serious off-pathway aggregation. The effects of 4 osmolytes--dimethylsulphoxide, glycerol, proline, and sucrose--on the refolding and reactivation of guanidine-denatured aminoacylase were studied by measuring aggregation, enzyme activity, intrinsic fluorescence spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra, and circular dichroism (CD) spectra. The results show that all the osmolytes not only inhibit aggregation but also recover the activity of aminoacylase during refolding in a concentration-dependent manner. In particularly, a 40% glycerol concentration and a 1.5 mol/L sucrose concentration almost completely suppressed the aminoacylase aggregation. The enzyme activity measurements revealed that the influence of glycerol is more significant than that of any other osmolyte. The intrinsic fluorescence results showed that glycerol, proline, and sucrose stabilized the aminoacylase conformation effectively, with glycerol being the most effective. All 4 kinds of osmolytes reduced the exposure of the hydrophobic surface, indicating that osmolytes facilitate the formation of protein hydrophobic collapse. The CD results indicate that glycerol and sucrose facilitate the return of aminoacylase to its native secondary structure. The results of this study suggest that the ability of the various osmolytes to facilitate the refolding and renaturation of aminoacylase is not the same. A survey of the results in the literature, as well as those presented here, suggests that although the protective effect of osmolytes on protein activity and structure is equal for different osmolytes, the ability of osmolytes to facilitate the refolding of various proteins differs from case to case. In all cases, glycerol was found to be the best stabilizer and a folding aid.