The asymmetric ATPase cycle of the thermosome: elucidation of the binding, hydrolysis and product-release steps.
Using a combination of intrinsic fluorescence to report ATP-induced rearrangements, quenched-flow to measure ATP hydrolysis "on-enzyme" and optical methods to probe the kinetics of product release, we have begun to dissect the process of energy transduction in the thermosome, a type II chaperonin from Thermoplasma acidophilum. Stoichiometric measurements of ATP binding reveal the tight association of eight nucleotide molecules per hexa-decamer, implying the filling of only one ring owing to strong negative cooperativity. After binding, we show that these eight ATP molecules are hydrolysed over the next 50 s, after which hydrolysis slows down markedly during the establishment of the steady state in the ATPase reaction, demonstrating that the kinetic system is off-rate limited. Looking in more detail, this rapid first-turnover can be dissected into two phases; the first occurring with a half-time of 0.8 s, the second with a half-time of 14 s, possibly reflecting the differential behaviour of the four alpha and four beta subunits in a single thermosome ring. To investigate the post-hydrolytic events, we used two heat-stable enzyme-linked optical assays to measure the rate of evolution of ADP and of phosphate from the thermosome active site. Neither product showed a rapid dissociation phase prior to the establishment of the steady state, showing that both are released slowly at a rate that limits the cycle. These data highlight the importance of the highly populated thermosome/ADP/Pi complex in the molecular mechanism.
Bigotti
Maria Giulia
MG
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. g.bigotti@bristol.ac.uk
Bellamy
Stuart R W
SR
Clarke
Anthony R
AR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites; D018833:Chaperonins / Q000378:metabolism*; D005453:Fluorescence; D006868:Hydrolysis; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D013822:Thermoplasma / Q000378:metabolism*; D056405:Thermosomes
D019843:Archaeal Proteins; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D018833:Chaperonins; D056405:Thermosomes