Ha J H, McKay D B
Beckman Laboratories for Structural Biology, Department of Structural Biology, Stanford University School of Medicine, California 94305.
Biochemistry. 1994 Dec 6;33(48):14625-35. doi: 10.1021/bi00252a031.
Steady-state kinetic, pre-steady-state kinetic, and equilibrium binding measurements have been applied to determine the rate constants of individual steps of the ATPase cycle for the recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal 44 kDa ATPase fragment. At 25 degrees C, pH 7.0, in the presence of 75 mM KCl and 4.5 mM Mg2+, the measured association rate constants for MgATP approximately hsc70 and MgADP approximately hsc70 are (2.7 +/- 0.5) x 10(5) and (4.1 +/- 0.5) x 10(5) M-1 s-1, respectively, while the dissociation rate constants are 0.0114 (+/- 0.0002) and 0.0288 (+/- 0.0018) s-1, respectively. MgATP (Kd = 0.042 microM) therefore binds to hsc70 more tightly than MgADP (Kd = 0.11 microM). ADP release is inhibited by inorganic phosphate (Pi), suggesting that product dissociation is ordered with Pi released first and ADP second. The rate of chemical hydrolysis of ATP is 0.0030 (+/- 0.0003) s-1 for hsc70 and 0.0135 (+/- 0.0033) s-1 for the 44 kDa fragment. The rate of Pi release is 0.0038 (+/- 0.0010) s-1 for hsc70 and 0.0051 (+/- 0.0006) s-1 for the 44 kDa fragment. For the 44 kDa fragment, Pi release is the slowest step in the ATPase cycle, while for hsc70, Pi release and chemical hydrolysis of MgATP have similar rates; in both cases, ADP release is a relatively rapid step in the ATPase cycle.
稳态动力学、预稳态动力学和平衡结合测量已被用于确定重组牛70 kDa热休克同源蛋白及其氨基末端44 kDa ATP酶片段的ATP酶循环各个步骤的速率常数。在25℃、pH 7.0、存在75 mM KCl和4.5 mM Mg2+的条件下，测得的MgATP与hsc70以及MgADP与hsc70的缔合速率常数分别为(2.7±0.5)×10^5和(4.1±0.5)×10^5 M^-1 s^-1，而解离速率常数分别为0.0114(±0.0002)和0.0288(±0.0018) s^-1。因此，MgATP(Kd = 0.042 μM)比MgADP(Kd = 0.11 μM)与hsc70的结合更紧密。无机磷酸盐(Pi)抑制ADP释放，表明产物解离是有序的，首先释放Pi，其次释放ADP。hsc70的ATP化学水解速率为0.0030(±0.0003) s^-1，44 kDa片段的为0.0135(±0.0033) s^-1。hsc70的Pi释放速率为0.0038(±0.0010) s^-1，44 kDa片段的为0.0051(±0.0006) s^-1。对于44 kDa片段，Pi释放是ATP酶循环中最慢的步骤，而对于hsc70，Pi释放和MgATP的化学水解速率相似；在这两种情况下，ADP释放都是ATP酶循环中相对较快的步骤。
