Kinetics of interaction of C1 inhibitor with complement C1s.
Kinetics of interaction of C1 inhibitor with complement C1s.
复制标题
C1 抑制剂与补体 C1 相互作用的动力学。
DOI：
10.1021/bi00361a023
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Ingham,KC
中科院分区：
文献类型：
--
作者：
Lennick,M;Brew,SA;Ingham,KC
补体丝氨酸蛋白酶Cls被其唯一已知的抑制剂Cl抑制剂抑制的动力学已用多种方法测定。一种方法是用偶联于色原的合成底物连续监测酯水解活性的丧失，而另一种方法是在解离条件下通过高压尺寸排阻色谱法监测稳定（共价）复合物的形成。另外的方法采用荧光探针来跟踪双分子复合物的形成，但预期不能区分共价产物和非共价（可逆）中间体。在广泛的抑制剂浓度范围内，通过各种方法获得的速率常数之间有很好的一致性，表明非共价中间体不会累积到显着的程度。该反应为二级反应，在30 ℃时的双分子速率常数为6.0 × 104 M ~（-1）s ~（-1），与Ca ~（2+）无关，活化能为11.0kcal/mol。在存在肝素的情况下，速率增加高达35倍，肝素显示以相似的亲和力（Kd= 2.0-3.3 µ）与所有三种组分（酶、抑制剂和复合物）结合。荧光探针l，l/-双（苯胺基）-4，4/-双（萘）-8，8 '-二磺酸盐[bis（ANS）]与复合物结合，Kd= 0.26 µ，条件是单个组分对染料的亲和力很小，这与在复合物形成期间在蛋白质表面上产生一个或多个疏水结合位点一致。1在调节补体系统中发挥关键作用，因为它是已知唯一与活化的补体蛋白酶Clr和Cls反应的循环抑制剂（Sim等人，1979年; Ziccardi，1981年;库珀，1985年）。结果1：1复合物对热和SDS稳定，但可与羟胺解离，表明组分之间形成共价酯键（Harpel和库珀，1975）。尽管这些反应的动力学已经在两个不同的实验室中进行了研究，但是对于与Cls的反应，所报道的双分子速率常数之间存在40倍的差异（Sim等人，1980; Nilsson & Wiman，1983）。关于反应的机制也存在不确定性，特别是关于可逆缔合的中间体络合物的重要性，该中间体络合物随后转化为稳定的中间体络合物。
The kinetics of inhibitionof the complement serine protease, Cls, by its only known inhibitor, Cl inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activitywith a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtainedby the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appearsto be pure second order with a bimolecular rate constant of 6.0 X 104 M-1 s" 1 at 30 C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all threecomponents (enzyme, inhibitor, and complex) with similar affinity (Kd= 2.0-3.3 µ). The fluorescent probe l, l/-bis (anilino)-4, 4/-bi (naphthalene)-8, 8'-di-sulfonate [bis (ANS)] bound to the complex with Kd= 0.26 µ under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.Human Cl inhibitor (Cl-Inh) 1 performs a pivotal function in the regulation of the complement system in thatit is the only circulatinginhibitor known to react with the activated complement proteases, Clr and Cls (Sim et al., 1979; Ziccardi, 1981; Cooper, 1985). The resulting 1: 1 complexes are stable to heat and SDS but can be dissociated with hydroxylamine, suggesting the formation of a covalent ester linkage between the components (Harpel& Cooper, 1975). Although the kinetics of these reactions have been investigatedin two dif-ferent laboratories, there is a 40-fold difference between the bimolecular rate constants reported for the reaction with Cls (Sim et al., 1980; Nilsson & Wiman, 1983). There is also uncertainty as to the mechanism of the reaction, in particular with respect to the importance of a reversibly associated intermediate complex whose subsequent conversion to a stable