Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation.
Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation.
复制标题
DOI：
10.1016/s0021-9258(17)39228-1
复制
发表时间：
1985-08
期刊：
影响因子：
--
通讯作者：
E. Eccleston;J. Howard
中科院分区：
文献类型：
--
作者：
E. Eccleston;J. Howard
人蛋白酶抑制剂α 2-巨球蛋白（α 2 M）通过与甲胺反应而失活。反应位点是具有硫羟酸酯性质的蛋白质官能团。为了确定硫醇酯裂解和蛋白质失活之间的关系，测量甲胺掺入和硫醇释放的速率。正如亲核试剂与硫羟酸酯的协同反应所预期的，速率是相同的。此外，这两个速率都是一阶相对于甲胺和二阶整体。通过测量总蛋白酶结合能力的损失来确定α 2 M的甲胺失活。该速率比硫羟酸酯裂解慢，并且具有显著的初始滞后。然而，失活遵循相同的时间过程作为构象变化的α 2 M，通过荧光染料结合，紫外差光谱，和有限的蛋白水解。因此，α 2 M的甲胺失活是一个连续的两步过程，其中硫醇酯裂解后是蛋白质构象变化。正是后者导致总蛋白酶结合能力的丧失。第二项试验用于监测甲胺对α 2 M的影响。该测定法测量对100 μ M大豆胰蛋白酶抑制剂灭活具有抗性的α 2 M结合蛋白酶的分数（小于50%）。与总蛋白酶结合能力相反，这个亚类消失的速率与甲胺裂解的硫醇酯一致。对高浓度大豆胰蛋白酶抑制剂具有抗性的α 2 M结合蛋白酶可能反映了随机交联到α 2 M的蛋白酶的部分。硫醇酯裂解和蛋白质构象变化速率均依赖于甲胺浓度。然而，硫羟酸酯的裂解依赖于甲胺作为亲核试剂的作用，而甲胺的离子强度则加速了构象变化。不裂解硫羟酸酯的其它盐和缓冲液增加了构象变化的速率。详细的动力学分析和模型的甲胺反应与α 2 M。利用甲胺反应研究α 2 M与蛋白酶结合的机制。在低离子强度下，蛋白质构象变化比甲胺引起的硫醇酯裂解慢得多。因此，在一些时间点，α 2 M的相当大部分具有所有四种硫醇酯裂解，但没有经历构象变化。这部分（约50%）保留了完整的蛋白酶结合能力。（400字处截断摘要）
The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)