Sohn Jungsan, Buhrman Gregory, Rudolph Johannes
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 2007 Jan 23;46(3):807-18. doi: 10.1021/bi061257y.
Using a combination of steady-state and single-turnover kinetics, we probe substrate association, dissociation, and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The rate constant for substrate association for the wild-type enzyme is 1.3 x 10(6) M(-1) s(-1). The rate constant for dissociation is slow compared to the rate constant for phosphate transfer to form the phospho-enzyme intermediate (k2 = 1.1 s(-1)), making Cdk2-pTpY/CycA a sticky substrate. Compared to the wild type, all hotspot mutants of residues at the remote docking site that specifically affect catalysis with the protein substrate (Arg488, Arg492, and Tyr497 on Cdc25B and Asp206 on Cdk2) have greatly slowed rate constants of association (70- to 4500-fold), and some mutants have decreased k2 values compared to that of the wild type. Most dramatically, R492L, despite showing no significant changes in a crystal structure at 2.0 A resolution, has an approximately 100-fold decrease in k2 compared to that of wild-type Cdc25B. The active site C473S mutant binds tightly to and dissociates slowly from Cdk2-pTpY/CycA (Kd = 10 nM, k(off) = 0.01 s(-1)). In contrast, the C473D mutant, despite showing only localized perturbations in the active site at 1.6 A resolution, has a much weaker affinity and dissociates rapidly (Kd of 2 microM, k(off) > 2 s(-1)) from the protein substrate. Overall, we demonstrate that the association of Cdc25B with its Cdk2-pTpY/CycA substrate is governed to a significant extent by the interactions of the remote hotspot residues, whereas dissociation is governed by interactions at the active site.
我们运用稳态动力学和单轮周转动力学相结合的方法，探究了Cdc25B磷酸酶与其Cdk2-pTpY/CycA蛋白底物反应中的底物结合、解离及化学反应过程。野生型酶的底物结合速率常数为1.3×10⁶ M⁻¹ s⁻¹。与磷酸转移形成磷酸化酶中间体的速率常数（k₂ = 1.1 s⁻¹）相比，解离速率常数较慢，这使得Cdk2-pTpY/CycA成为一种粘性底物。与野生型相比，在远距离对接位点特异性影响与蛋白底物催化作用的残基（Cdc25B上的Arg488、Arg492和Tyr497以及Cdk2上的Asp206）的所有热点突变体，其结合速率常数大幅减慢（70至4500倍），并且一些突变体与野生型相比k₂值降低。最显著的是，R492L尽管在2.0 Å分辨率的晶体结构中未显示出明显变化，但其k₂值与野生型Cdc25B相比降低了约100倍。活性位点C473S突变体与Cdk2-pTpY/CycA紧密结合且解离缓慢（Kd = 10 nM，k(off) = 0.01 s⁻¹）。相比之下，C473D突变体尽管在1.6 Å分辨率下仅在活性位点显示局部扰动，但其与蛋白底物的亲和力弱得多且解离迅速（Kd为2 μM，k(off) > 2 s⁻¹）。总体而言，我们证明Cdc25B与其Cdk2-pTpY/CycA底物的结合在很大程度上受远距离热点残基相互作用的支配，而解离则受活性位点相互作用的支配。
