Ghosh Kaushik, Pal Atasi, Chattopadhyaya Rajagopal
Department of Biochemistry, Bose Institute, P-1/12, CIT Scheme VII M, Calcutta 700054, India.
Biochem J. 2004 Apr 15;379(Pt 2):325-30. doi: 10.1042/BJ20031834.
The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degrees C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the lambda repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans -cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans -cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.
在本研究中，分别于27℃、37℃和42℃下，在pH 10.6的条件下测定了λ cI阻遏蛋白不依赖RecA的、自发的、pH依赖性自切割的一级速率常数。阻遏蛋白的自切割在pH 9和8时也会发生，不过速率逐渐变慢。我们证明，自发自切割在与操纵基因结合的状态下也会发生，其速率高于或等于溶液中的速率，这取决于pH值。由于在无操纵基因和与操纵基因结合的阻遏蛋白中速率常数几乎相等，可以推断在中性pH条件下，两种形式的切割位点相对于催化位点具有相似的结构和动力学。使用大大过量的苯甲基磺酰氟（PMSF）进行共价修饰会抑制λ阻遏蛋白的自切割。利用我们最近的λ阻遏蛋白模型解释了获得这种共价修饰的困难。之前观察到缺乏关键催化丝氨酸或赖氨酸残基的突变型LexA阻遏蛋白会发生双分子II型反式切割[Kim和Little（1993年），《细胞》（马萨诸塞州剑桥）73卷，1165 - 1173页]，但它仍然可以被具有改进或超可切割特性且没有自身切割位点的85 - 202“酶”片段切割。当使用共价修饰的完整λ阻遏蛋白作为底物以及纯化的野生型λ阻遏蛋白112 - 236片段作为“酶”时，未观察到这种II型反式切割。所有这些结果表明，对于野生型λ阻遏蛋白，在无操纵基因和与操纵基因结合的状态下，催化位点都靠近切割位点。在裂解途径中，阻遏蛋白主要通过RecA介导的切割被切割，其发生速度比自发自切割快得多；这种缓慢、自发但持续的自切割的可能生物学意义与溶原状态有关，此时不存在RecA介导的切割。
