title: P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).
abstract: mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Emily V
authors.initials: EV
authors.lastname: Wong
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Cao
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
authors.forename: Judit
authors.initials: J
authors.lastname: Vörös
authors.affiliation: Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Monique
authors.initials: M
authors.lastname: Merchant
authors.affiliation: Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Yorgo
authors.initials: Y
authors.lastname: Modis
authors.affiliation: Department of Biological Sciences and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Hackney
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, AB, T6G 2H7, Canada. Electronic address: ben.montpetit@ualberta.ca.
authors.forename: Ben
authors.initials: B
authors.lastname: Montpetit
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA. Electronic address: enrique.delacruz@yale.edu.
authors.forename: Enrique M
authors.initials: EM
authors.lastname: De La Cruz
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D007700:Kinetics; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D029543:Nucleocytoplasmic Transport Proteins; D010710:Phosphates; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D000251:Adenosine Triphosphatases; C492382:DBP5 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
keywords: RNA helicase; kinetics; mRNA export; mantATP; thermodynamics