Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding
Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding
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DOI：
10.1074/jbc.m000342200
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发表时间：
2000-07-14
影响因子：
4.8
通讯作者：
Coluccio, LM
中科院分区：
文献类型：
--
作者：
Geeves, MA;Perreault-Micale, C;Coluccio, LM
MI1IQ是一种钙调蛋白复合体和一个表位标记的85 kDa片段，它代表了哺乳动物肌球蛋白I基因大鼠MYR-1(130-kDa肌球蛋白I或MI130)的6个钙调蛋白结合IQ结构域中的第一个。通过检测MIIQ的性质，我们已经确定了决定哺乳动物肌球蛋白I的ATP水解周期的瞬时动力学参数。瞬时动力学表明，MIIQ对肌动蛋白的亲和力为12 nM。ATP诱导的肌动蛋白-MIIQ解离是双相的。快相依赖于[ATP]，而慢相不依赖于[ATP]；两个相都表现出对钙的敏感性。加入ADP可消除快相，在有钙离子存在的情况下，效果半饱和需要10µM，而在无钙离子的情况下，需要3muM ADP。慢相与腺苷二磷酸的释放具有相同的速率常数(在有钙和无钙时分别为8和3 S(-1))，但不能通过降低[腺苷二磷酸]来消除。我们对这些结果的解释是，肌动蛋白-肌球蛋白I在平衡状态下以两种形式存在，其中一种不能与核苷酸结合。这些结果还表明，MYR-1的COOH末端5个钙调蛋白结合域的缺失并不影响MI130的动力学性质，动力学的钙敏感性很可能是由于钙与第一IQ结构域结合所致。
MI1IQ is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI130). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI1IQ. Transient kinetics reveal that the affinity of MI1IQ for actin is 12 nM. The ATP-induced dissociation of actin-MI1IQ is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca2+ sensitivity. The fast phase is eliminated by the addition of ADP, 10 mu M being required for half-saturation of the effect in the presence of Ca2+ and 3 mu M ADP in the absence of Ca2+. The slow phase shares the same rate constant as ADP release (8 and 3 s(-1) in the presence and absence of Ca2+, respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI130 and that the Ca2+ sensitivity of the kinetics are in all likelihood due to Ca2+ binding to the first IQ domain.