E COLI CHAPERONIN: ATP HYDROLYSIS KINETICS BY TWO RING BIOCALORIMETRY

一种新的，通用的方法来测量酶的活性， 微量量热法（托德和戈麦斯，1997）应用于E.杆菌 伴侣蛋白GroEL。 生物量热分析测量的是 在底物转化为产物的过程中的演变。 量热法因此 给出了酶促反应速率的直接测量，如 与其中速率被计算为 量对时间的导数。 等温量热分析 GroEL ATP酶活性的高分辨率分析， 周转 在ATP再生系统存在下， 是双相的（即，两个环的ATP水解动力学 可以区分）。 两个环中的一个非常容易受到 产物抑制，因此在没有再生系统的情况下， 仅观察到两个环中的一个的翻转。 的影响 离子辅因子K+和Mg 2+，以及未折叠蛋白（cam a-lac）， 并研究了 两个环的动力学变得难以区分 在高[K+]，而动力学分离良好，在较低 浓度的 GroEL可以结合多达两个未折叠的蛋白质（一个在 每个环）。 一个或两个未折叠的ATP酶活性 检查结合的蛋白质。

A novel, universal method for measuring enzymatic activity using micro-calorimetry (Todd & Gomez, 1997) is applied to the E. coli chaperonin GroEL. Biocalorimetric assays measure the rate of heat evolution during conversion of substrate to product. Calorimetry thus gives a direct measurement of the rate of an enzymatic reaction, as opposed to conventional assays where rate is calculated as a derivative of a quantity with time. Isothermal calorimetric analysis of GroEL ATPase activity allows high-resolution rate analysis of turnover. In the presence of ATP regenerating system, the kinetics were biphasic (i.e., the kinetics of ATP hydrolysis by the two toroids can be distinguished). One of the two rings is highly susceptible to product inhibition, thus in the absence of a regenerating system, turnover by only one of the two rings is observed. The effects of ionic cofactors K+ and Mg2+, and unfolded proteins (cam a-lac) were also studied. The kinetics of the two rings become indistinguishable at high [K+], whereas the kinetics are well-separated at lower concentrations. GroEL can bind up to two unfolded proteins (one on each ring). The ATPase activity with one or two unfolded proteins bound is examined.