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.
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.
Málnási-Csizmadia
A
A
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK.
Pearson
D S
DS
Kovács
M
M
Woolley
R J
RJ
Geeves
M A
MA
Bagshaw
C R
CR
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003080:Cold Temperature; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics*; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics*; D011312:Pressure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D020409:Molecular Motor Proteins; C016952:N-acetyltryptophanamide; D014364:Tryptophan; D000255:Adenosine Triphosphate; D024462:Myosin Type II