Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.
Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.
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DOI：
10.1073/pnas.84.12.3987
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发表时间：
1987-06
影响因子：
11.1
通讯作者：
S. N. Slilaty;John W. LITTLEtt
中科院分区：
文献类型：
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作者：
S. N. Slilaty;John W. LITTLEtt
大肠杆菌的LexA抑制因子在体内通过需要激活RecA蛋白的特定裂解反应失活。在体外，裂解需要在中性pH下激活RecA，在碱性pH下自发进行。这两种裂解反应具有相似的特异性，表明RecA间接刺激自裂解，而不是直接作为蛋白酶。我们根据动力学数据、与其他可切割抑制因子的同源性以及与蛋白酶的潜在相似性，利用定点诱变技术改变了LexA中选定的氨基酸残基，研究了裂解的化学机制。丝氨酸-119和赖氨酸-156被转化为丙氨酸，这是一种具有非活性侧链的残基，导致两个突变蛋白具有正常的抑制功能和表面上正常的结构，但在两种类型的裂解反应中都完全缺乏。丝氨酸-119也被转化为半胱氨酸，这是另一种具有亲核侧链的残基，导致蛋白质以显著的速度被切割。这些和其他观察结果表明，可剪切肽键的水解机制与丝氨酸蛋白酶类似，丝氨酸-119是亲核试剂，赖氨酸-156是激活剂。讨论了RecA可能扮演的角色。
LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein. In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease. We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases. Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction. Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate. These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator. Possible roles for RecA are discussed.