利用CD和FTIR研究氨基酰化酶的Holo-酶和Apo-酶的二级结构

利用去卷积Fourier变换红外光谱和圆二色光谱的方法研究了在Zn~(2+)存在和缺失两种情况下,该酶的二级结构的变化情况。结果表明,Zn~(2+)的存在与否对酶的二级结构产生了显著的影响。天然酶脱去Zn~(2+)后,其有序度明显降低,说明酶的活性部位含有相对较多的有序结构,Zn~(2+)对活性部位及活性部位附近的构象起到了一定的稳定作用。     

氨基酰化酶在脲溶液中变性时构象变化速度与失活速度的比较

应用了邹氏不可逆抑制动力学的方法,在变性剂存在的条件下,连续监测酶催化的底物反应过程,测定了氨基酰化酶在不同浓度脲溶液中失活的速度常数,并考察了底物的存在对酶失活的保护作用。同时,还比较了氨基酰化酶在不同浓度脲溶液中变性时失活和构象变化的速度。1mol/L,2mol/L脲变性时失活速度常数为为1.62×10~(-2)S~(-1)和2.05×10~(-2)S~(-1),但是酶分子的整体构象尚未发生明显变化。3mol/L脲变性时,失活速度常数为2.56×10~(-2)S~(-1),而变性速度常数为3.72×10~(-3)S~(-1)。失活速度比构象变化速度快大约一个数量级。在4mol/L,5mol/L,6mol/L脲溶液中变性时,酶分子快速失活,而其构象变化速度常数分别为5.27×10~(-3)S~(-1)。5.47×10~(-3)S~(-1),5.56×10~(-3)S~(-1)。可见,在相同浓度的脲溶液中,氨基酰化酶的失活速度明显快于酶分子整体构象变化的速度。上述结果表明,含有辅基金属离子Zn~(2+)的氨基酰化酶的活性部位较酶分子的整体结构也具有一定的柔性。     

肌酸激酶在胍溶液中的变性与失活速度的比较

本文以紫外差光谱、荧光光谱为监测手段测定肌酸激酶的变性与失活过程均为一级反应。在3M胍溶液中,30℃测得的失活速度常数为5.9秒~(-1),变性速度常数为1.9秒~(-1)。1M胍变性时,失活速度常数为4.3秒~(-1),而交性速度常数明显降低为0.04秒~(-1)。0.5M胍变性时,失活为两个一级过程,其速度常数分别为3.6秒~(-1)与0.003秒~(-1),此外酶还保持有3.4％的剩余活力;变性速度常数为0.004秒~(-1)。在0.3M胍中,酶的紫外差吸收及荧光变化都很小,但是60％的活力仍然迅速丧失,最后还保持有30％剩余活力。上述结果表明,酶的活性部位可能比较脆弱。酶分子双体的解聚或盐酸胍的抑制作用也可能引起酶的快速失活。低浓度胍变性时,失活过程的多相性意味着可能存在有部分活力的中间态。     

Kinetics of Inactivation of Aminoacylase I During Modification of Its Thiol Groups by DPDS and PCMB

The kinetics theory of the substrate reaction during modification of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of aminoacylase I by DPDS and PCMB.From the results obtained we have found that the inactivation reaction of aminoacylase I by DPDS is noncomplexing inhibition,and PCMB reaction is complexing inhibition.The microscopic constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined.     

DTNB标记的肌酸激酶的水解及氰解反应的研究

本文研究了DTNB标记的肌酸激酶(S,S′-二-TNB-CK)氰解反应,发现在Degani等人的氰解反应的条件下,同时有水解反应存在。当没有KCN存在时,在pH9.5的条件下,DTNB标记的CK可以迅速地被水解,并释放出TNB基团。水解反应呈双相的一级反应,在快相反应中每个酶分子大约释放一个TNB基团。当水解后的产物进一步与KCN反应时,每个酶分子大约又释放一个TNB基团,反应则呈单相的一级反应。上述结果表明在DTNB修饰酶中,两个被修饰的巯基分别处于二聚体肌酸激酶的每一个亚基的活性部位上,其中一个TNB基团在水解反应中迅速被释放出来而另一个以很慢的速度反应并很难反应完全。而水解产物进行氰解时,那个很难被水解的TNB基团此时被释放出来。这表明了肌酸激酶的两个亚基结合是不对称的,因而导致两个活性部位的巯基处于不同的化学微环境中。此外还发现在水解过程或氰解过程中伴随着TNB基团的释放,活性部位部分巯基被还原成游离巯基,同时活力也得到部分恢复并呈正相关作用。这进一步说明CK中可反应的半胱氨酸巯基是酶的必需基因,并处于酶分子的活性部位上。     

平面阴离子对肌酸激酶活性的抑制动力学研究

本文将邹氏不可逆改变动力学理论与方法运用于平面阴离子对双底物的肌酸激酶的慢可逆抑制作用的研究中,根据理论分析和实验结果,推导并简化了动力学方程,定量地测定了抑制作用的微观动力学常数,探讨了平面阴离子对肌酸激酶的抑制作用机制。     

DPDS和PCMB对氨基酰化酶Ⅰ不可逆抑制动力学研究

本文运用邹氏酶活性不可逆改变的动力学,研究了巯基试剂DPDS,PCMB对氨基酰化酶Ⅰ活性的不可逆抑制作用,判明了这些-SH试剂对于氨基酰化酶Ⅰ的反应类型,发现DPDS对氨基酰化酶Ⅰ的反应为非络合型反应,而PCMB对氨基酰化酶Ⅰ的反应则为络合型,同时,还测定了DPDS,PCMB对氨基酰化酶Ⅰ的不可逆抑制作用的微观速度常数。     

QUANTITATIVE RELATION BETWEEN MODIFICATION OF FUNCTIONAL GROUPS OF PROTEINS AND THEIR BIOLOGICAL ACTIVITY——A COMPUTER PROGRAM FOR ASCERTAINING THE NUMBER OF ESSENTIAL RESIDUES

Based on a statistical treatment of the quantitative relation between the extents of mod-ification of protein functional groups and the decrease of their biological activity, a meth-od was proposed (Tsou, Sci. Sin., 1962, 11:1535) and widely used for the determination ofthe number of residues essential for the biological activity ot the protein modified. How-ever, the original method depends on a trial and error approach and manual calculation tofind the best fit for the data obtained. A computer program is described in this paper forthe treatment of experimental data to produce the number of essential and non-essentialgroups modified and the ratio of rates for their modification which best fit the data ob-tained. Applications of this method show that in some cases wrong conclusions were reachedin literature and in other cases quantitative conclusions can sometimes be drawn when orig-inally either no quantitative data treatment was presented or attempts at manual curve fit-ting were unsuccessful.     

A COMPARISON OF DENATURATION AND INACTIVATION RATES OF CREATINE KINASE IN GUANIDINE SOLUTIONS

Both the denaturation, as followed by UV absorbance and fluorescence changes, and inac-tivation of creatine kinase in guanidine solutions have been found to be first order reactions.In 3 M guanidine, at 30℃, the inactivation rate constant was found to be 5.9 sec~(-1) and thedenaturation rate constant 1.9 sec~(-1). At lower guanidine concentrations, the inactivation rateconstants were only little affected whereas the denaturation rate constants decreased markedly,being of the order of 0.04 in 1 M and 0.004 in 0.5 M guanidine. The kinetics of the inactiva-tion reaction in 0.5 M guanidine was found to be in agreement with a combination of two firstorder reactions. The enzyme lost activity first by a fast reaction with a rate constant onlyslightly lower than the rate constant in 3 M guanidine followed by a slower reaction with a rateconstant of 0.003 sec~(-1). In 0.3 M guanidine, very little change in either UV absorbance or influorescence was observed, but, in sharp contrast, the enzyme lost considerable activity by a fastreaction and this was followed by a slower reaction of inactivation. Even after prolongeddenaturation in 0.5 and 0.3 M guanidine, residual activities of 3.4% and 30% remained res-pectively. The above results suggest a very fragile active site although dissociation of thedimer and reversible guanidine inhibition may also contribute to the initial rapid inactiva-tion. It is also to be noted that the multiphasic courses of inactivation at lower guanidineconcentrations seem to suggest the presence of partly active intermediates during denaturation.     

氧化型肌酸激酶的研究

利用NR/R双向对角线SDS-PAGE作者首次将过去被认为均一的兔肌肌酸激酶分离为含有链内二硫键的和不含任何二硫键的两种肌酸激酶(前者称为“氧化型肌酸激酶”,后者称为“还原型肌酸激酶”)。它们具有相同的化学组成,相同的亚基分子量,相同的等电点及几乎相同的催化活性,因此用一般常用的分离纯化手段很难将它们分离开来。进一步的研究表明,氧化型的肌酸激酶也含有一对表面巯基,并对酶的活性同样是必需的。氧化型肌酸激酶分子中,每一个亚基含有一个链内二硫键,在没有变性的条件下,不能被DTT还原,而还原型肌酸激酶通过某些氧化剂氧化可以形成这个二硫键,因此作者认为该二硫键可能是由内埋较浅的一对巯基氧化而成的。