Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue

Malnasi-Csizmadia, A. and Pearson, D.S. and Kovacs, M. and Woolley, R.J. and Geeves, M.A. and Bagshaw, C.R. (2001) Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue. Biochemistry, 40 (42). pp. 12727-12737. ISSN 0006-2960 . (The full text of this publication is not available from this repository)

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Abstract

The fluorescence emission intensity from a conserved tryptophan residue (W501) located in the relay loop (F466 to L516) of the Dicytostelium discoideum myosin II motor domain is sensitive to ATP binding and hydrolysis. The initial binding process is accompanied by a small quench in fluorescence, and this is followed by a large enhancement that appears coincident with the hydrolysis step. Using temperature and pressure jump methods, we show that the enhancement process is kinetically distinct from but coupled to the hydrolysis step. The fluorescence enhancement corresponds to the open-closed transition (k(obs) approximately 1000 s(-1) at 20 degrees C). From the overall steady-state fluorescence signal and the presence or absence of a relaxation transient, we conclude that the ADP state is largely in the open state, while the ADP.AlF(4) state is largely closed. At 20 degrees C the open-closed equilibria for the AMP.PNP and ADP.BeF(x) complexes are close to unity and are readily perturbed by temperature and pressure. In the case of ATP, the equilibrium of this step slightly favors the open state, but coupling to the subsequent hydrolysis step gives rise to a predominantly closed state in the steady state. Pressure jump during steady-state ATP turnover reveals the distinct transients for the rapid open-closed transition and the slower hydrolysis step.

Item Type: Article
Additional information: 0006-2960 (Print) Journal Article Research Support, Non-U.S. Gov't
Uncontrolled keywords: Adenosine Triphosphate/*metabolism Animals Cold Dictyostelium/*chemistry/*genetics Heat Hydrolysis Kinetics Molecular Motor Proteins/chemistry/genetics Mutagenesis, Site-Directed Myosin Type II/*chemistry/*genetics Pressure Protein Conformation Protein Structure, Tertiary/genetics Spectrometry, Fluorescence/instrumentation/methods Tryptophan/*analogs & derivatives/*chemistry/*genetics/metabolism
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: Michael Geeves
Date Deposited: 29 Aug 2008 11:19
Last Modified: 14 Jan 2010 14:13
Resource URI: http://kar.kent.ac.uk/id/eprint/3860 (The current URI for this page, for reference purposes)
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