Grossman S H, France R M, Mattheis J R
Department of Chemistry, University of South Florida, Tampa 33620.
Biochim Biophys Acta. 1992 Sep 4;1159(1):29-36. doi: 10.1016/0167-4838(92)90071-k.
(1) A single subunit and both subunits of creatine kinase from rabbit muscle was derivatized at the active site with the thiol-specific reagent 2-(4'-(iodoacetamido)anilino)-naphthalene-6-sulfonic acid. (2) The highly biphasic kinetics of the labelling reaction were characterized from measurements of activity, steady-state fluorescence and anisotropy. Derivatization of one thiol and both thiols resulted in 48 and 100% inhibition, respectively. The dead-end complex (DEC), consisting of creatine, MgADP and protein, inhibited the rate, but not the extent, of derivatization and resulted in a 2-fold increase in fluorescence. (3) The fluorescence of singlylabelled (1AANS/CK) and doublylabelled (2AANS/CK) protein exhibited three discrete lifetime components or a two-term Lorentzian distribution. The decay laws for both preparations were not remarkably different, except that, unlike 1AANS/CK, the longer decay component of 2AANS/CK was distributed, which narrowed in the presence of the DEC. (4) The steady-state anisotropies of 1AANS/CK and 2AANS/CK at 25 degrees C were 0.305 and 0.240, respectively. It was concluded that the fast reacting site was immobile and the slow reacting site was flexible. Kinetics of labelling and anisotropy emission spectra indicated that the DEC immobilized the flexible site. (5) The anisotropy decay of 1AANS/CK with and without the DEC was described by a rotational correlation time of about 50 ns, characteristic of the molecular rotation of the CK dimer. At least two terms were required to fit the data for 2AANS/CK, indicating additional segmental motion which was eliminated upon formation of the DEC. (6) Energy transfer from tryptophans to AANS indicated movement of approx. 3 A accompanying formation of the DEC.
(1) 兔肌肉肌酸激酶的单个亚基和两个亚基在活性位点用硫醇特异性试剂2-(4'-(碘乙酰胺基)苯胺)-萘-6-磺酸进行衍生化。(2) 通过活性、稳态荧光和各向异性的测量对标记反应的高度双相动力学进行了表征。一个硫醇和两个硫醇的衍生化分别导致48%和100%的抑制。由肌酸、MgADP和蛋白质组成的终产物复合物(DEC)抑制了衍生化的速率，但不影响其程度，并导致荧光增加2倍。(3) 单标记(1AANS/CK)和双标记(2AANS/CK)蛋白质的荧光表现出三个离散的寿命成分或双项洛伦兹分布。两种制剂的衰变规律没有显著差异，只是与1AANS/CK不同，2AANS/CK的较长衰变成分呈分布状态，在DEC存在时变窄。(4) 1AANS/CK和2AANS/CK在25℃时的稳态各向异性分别为0.305和0.240。得出的结论是，快速反应位点是固定的，而慢速反应位点是灵活的。标记动力学和各向异性发射光谱表明DEC使灵活位点固定。(5) 有和没有DEC时1AANS/CK的各向异性衰变由约50 ns的旋转相关时间描述，这是CK二聚体分子旋转的特征。至少需要两项来拟合2AANS/CK的数据，表明存在额外的片段运动，在DEC形成时这种运动被消除。(6) 从色氨酸到AANS的能量转移表明，在DEC形成时伴随有约3 Å的移动。
