Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease.
Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease.
复制标题
精液核糖核酸酶半胱氨酸残基 31 和 32 超反应性的分子基础。
DOI：
10.1021/bi00326a005
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
G. D'alessio
中科院分区：
文献类型：
--
作者：
A. Parente;B. Merrifield;G. Geraci;G. D'alessio
通过研究单体酶中与相邻半胱氨酸-31和-32的二硫化物的快速反应动力学，研究了二聚牛精液核糖核酸酶的位置31和32处的亚基间二硫化物对还原剂的高反应性的分子基础，通过选择性还原亚基间二硫化物暴露。带负电荷的和中性的二硫化物试剂用于测量在中性pH值的硫醇反应速率。作为pH值的函数的动力学研究允许我们定义感兴趣的硫醇的pK值，并表示通过测量与二硫化物试剂的SH基团的反应动力学间接确定蛋白质中的SH基团的pK值的可能性。结果进行了比较与合成硫醇肽和模型化合物在相同的条件下得到的那些。这些数据表明，精液核糖核酸酶的亚基间二硫化物的超反应性与相邻半胱氨酸残基31和32在中性pH下的高反应性相匹配，与所有测试的小硫醇化合物相比。精液核糖核酸酶Ac-Met-Cys-Cys-Arg-Lys-Met-OH的合成六肽片段，包括两个感兴趣的半胱氨酸残基，甚至更具反应性。这些数据，以及本文报道的其他结果，得出的结论是，牛精液核糖核酸酶的位置31和32的半胱氨酸残基在中性pH下的超反应性主要取决于附近存在的带正电荷的基团，特别是赖氨酸-34的ε-NH 2，并受到两个巯基的相邻性和蛋白质三级结构的影响。
The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.