Identification of a major inter-ring coupling step in the GroEL reaction cycle.
It has been shown previously that the double-ring structure of GroEL can be converted to a single-ring species by site-directed amino acid replacements at the ring interface and that the resultant molecule retains many of the crucial chaperonin properties; it is structurally stable, hydrolytically active, and can bind both the co-chaperonin, GroES, and unfolded substrate proteins. By comparing the behavior of the double- and single-ring structures in response to nucleotide binding and hydrolysis, we elucidate steps in the ATP-driven reaction cycle at which there is conformational coupling between the rings. Remarkably, the parting of the rings has little effect either on the thermodynamic properties of ATP binding or on the ATP-induced conformational changes prior to hydrolysis. However, there is a marked effect on the rate-limiting process in the steady-state cycle; a step that is coincident with bond cleavage in ATP. The effect of the ring-ring interaction is to increase its activation enthalpy from 42.0 to 94.2 kJ/mol. These results show that the major conformational coupling step, where structural rearrangements in one ring are propagated to the other, is the slowest process the ATPase cycle of GroEL.
Poso
Daniel
D
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. d.poso@bristol.ac.uk
Clarke
Anthony R
AR
Burston
Steven G
SG
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000495:Allosteric Site; D018834:Chaperonin 60 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005470:Fluorometry; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D018834:Chaperonin 60; D010710:Phosphates; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases