通过 ADP-A1F4 位点特异性抑制揭示不对称 Rep 二聚体 P2S 水解 ATP 的双位点机制。

A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-A1F4.

作者信息

Wong I, Lohman T M

机构信息

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

出版信息

Biochemistry. 1997 Mar 18;36(11):3115-25. doi: 10.1021/bi9621977.

DOI:10.1021/bi9621977

PMID:9115987

Abstract

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction--the asymmetric Rep dimer state, P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-A1F4. We found that when ADP-A1F4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s(-1) per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-A1F4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP <==> E-ATP <==> E'-ATP <==> E'-ADP-Pi <==> E-ADP-Pi <==> E-ADP + Pi <==> E + ADP + Pi, with K1 = (2.0 +/- 0.85) x 10(5) M(-1), k2 = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K3 = 4.0 +/- 0.4 (k3 > 200 s(-1)), k4 = 1.2 +/- 0.14 s(-1), k(-4) << 1.2 s(-1), K5 = 1.0 +/- 0.2 mM, and K6 = 80 +/- 8 microM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.

摘要

大肠杆菌Rep解旋酶是一种二聚体马达蛋白，它利用ATP结合和水解所产生的能量，催化双链DNA瞬时解旋形成单链（ss）DNA。为了理解这种能量转导机制，我们采用稳态前方法研究了DNA解旋反应中一个重要中间体——不对称Rep二聚体状态P2S的ATP结合和水解动力学，在该状态下，ss DNA [dT(pT)15]仅与Rep二聚体的一个亚基结合。为了区分二聚体中固有的两个潜在ATP酶活性位点，我们构建了一个亚基与ss DNA共价交联的二聚体，其中一个或另一个ATP酶位点选择性地与紧密结合的过渡态类似物ADP - A1F4复合。我们发现，当ADP - A1F4从与ss DNA结合的亚基反式结合到Rep亚基上时，每个二聚体的稳态ATP酶活性恢复到18 s⁻¹（相当于野生型P2S）。然而，当ADP - A1F4和ss DNA都结合到同一个亚基（顺式）上时，会观察到一个可滴定的ATP水解爆发，对应于ATP的单次周转。采用快速化学淬灭流动技术解析了顺式二聚体未结合配体的Rep亚基ATP水解的以下最小机制：E + ATP ⇌ E - ATP ⇌ E' - ATP ⇌ E' - ADP - Pi ⇌ E - ADP - Pi ⇌ E - ADP + Pi ⇌ E + ADP + Pi，其中K1 = (2.0 ± 0.85) × 10⁵ M⁻¹，k2 = 22 ± 3.5 s⁻¹，k⁻² < 0.12 s⁻¹，K3 = 4.0 ± 0.4（k3 > 200 s⁻¹），k4 = 1.2 ± 0.14 s⁻¹，k⁻⁴ << 1.2 s⁻¹，K5 = 1.0 ± 0.2 mM，K6 = 80 ± 8 μM。该机制的一个显著特征是存在一个动力学捕获的长寿命紧密核苷酸结合状态E' - ADP - Pi。在我们关于进行性DNA解旋过程中Rep二聚体易位的“亚基切换”模型[Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411 - 422]的背景下，这种状态可能起到能量储存的作用，使ATP结合和水解产生的能量能够被利用并储备起来用于DNA解旋。