Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine , Reno, Nevada 99557, United States.
Biochemistry. 2013 Nov 26;52(47):8489-500. doi: 10.1021/bi401001x. Epub 2013 Nov 11.
During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.
在平滑肌收缩的激活过程中，一个肌球蛋白轻链激酶（MLCK）分子迅速磷酸化许多平滑肌肌球蛋白（SMM）分子，这表明肌肉激活速率受 MLCK-SMM 相互作用的动力学影响。为了确定 MLCK 激活 SMM 的限速步骤，我们在体外系统中测量了钙钙调蛋白（Ca²⁺CaM）-MLCK 介导的 SMM 磷酸化的动力学和相应的 SMM 基于 F-肌动蛋白运动的起始，其中 SMM 附着在盖玻片表面上。将 SMM 磷酸化的时间过程拟合到动力学模型中，得到初始磷酸化速率 kp(o)约为 1.17 个头部 s⁻¹ MLCK⁻¹。此外，我们使用全内反射荧光显微镜测量了与表面附着的 SMM 和磷酸化 SMM 相互作用的单个链霉亲和素包被的量子点标记的 MLCK 分子的停留时间。从这些数据中，从磷酸化 SMM 上的解离速率常数为 0.80 s⁻¹，与上面提到的 kp(o)相似，并且与溶液中的速率测量值相似。该离解速率基本上与 SMM 的磷酸化状态无关。根据我们测量的离解速率和 Kd 值以及肌肉中 SMM 和 MLCK 浓度的估算，我们预测 MLCK 从磷酸化 SMM 的解离是限速的，并且磷酸化步骤的速率快于该解离速率。此外，与 SMM 的结合（11-46 s⁻¹）比与 pSMM 的结合（<0.1-0.2 s⁻¹）快得多。这表明 MLCK 与未磷酸化的 SMM 相互作用的概率比与磷酸化的 SMM 相互作用的概率高 55-460 倍。这将避免 MLCK 与先前磷酸化的 SMM 发生无益的相互作用，从而潜在地导致肌肉中磷酸化的更快速率。
