Eckhardt U, Hanstein W G
Institute for Physiological Chemistry, Ruhr-University Bochum, Germany.
Biochim Biophys Acta. 1993 Oct 4;1144(3):419-25. doi: 10.1016/0005-2728(93)90129-4.
Two ATP analogs, 2- and 8-azidoadenyl-5'-yl imidodiphosphate, were synthesized, purified and utilized as inhibitors of soluble beef heart mitochondrial F1-ATPase under non-photolytical conditions. In the range of 5 microM to 3 mM ATP, the initial rates of ATP hydrolysis in the presence and absence of the inhibiting ATP analogs can be adequately described by two pairs of Km and Vmax values (3 microM, 8.5 mumol ATP/min per mg; 255 microM, 42.0 mumol ATP/min per mg). With increasing inhibitor concentrations, the apparent Km,2 increases as in competitive inhibition, while Vmax,1 decreases as in non-competitive inhibition. The Ki values derived for both types of inhibition are similar, but strongly different for 2- and 8-azido-AMP-PNP (4 microM and 460 microM, respectively). The decrease of the high-affinity Vmax is compensated by an increase in low-affinity catalysis, resulting in a constant sum of maximal velocities. These data can be described by a model where two sites interact with negative cooperativity in binding of substrate.
合成、纯化了两种ATP类似物，即2-叠氮腺苷-5'-亚氨基二磷酸和8-叠氮腺苷-5'-亚氨基二磷酸，并在非光解条件下用作可溶性牛心线粒体F1-ATP酶的抑制剂。在5微摩尔至3毫摩尔ATP范围内，存在和不存在抑制性ATP类似物时ATP水解的初始速率可用两对Km和Vmax值（3微摩尔，8.5微摩尔ATP/分钟/毫克；255微摩尔，42.0微摩尔ATP/分钟/毫克）充分描述。随着抑制剂浓度增加，表观Km,2如竞争性抑制那样增加，而Vmax,1如非竞争性抑制那样降低。两种抑制类型的Ki值相似，但2-叠氮-AMP-PNP和8-叠氮-AMP-PNP的Ki值差异很大（分别为4微摩尔和460微摩尔）。高亲和力Vmax的降低由低亲和力催化的增加来补偿，导致最大速度总和恒定。这些数据可用一个模型来描述，其中两个位点在底物结合中以负协同性相互作用。
