Setting the chaperonin timer: The effects of K+ and substrate protein on ATP hydrolysis
Abstract
The effects of potassium ion on the nested allostery of GroEL are due to increases in the affinity for nucleotide. Both positive allosteric transitions, TT-TR and TR-RR, occur at lower [ATP] as [K+] is increased. Negative cooperativity in the double-ringed system is also due to an increase in the affinity of the trans ring for the product ADP as [K+] is increased. Consequently, (i) rates of ATP hydrolysis are inversely proportional to [K+] and (ii) the residence time of GroES bound to the cis ring is prolonged and the hemicycle time extended. Substrate protein suppresses negative cooperativity by decreasing the affinity of the trans ring for ADP, reducing the hemicycle time to a constant minimum. The trans ring thus serves as a variable timer. ATP added to the asymmetric GroEL-GroES resting-state complex lacking trans ring ADP is hydrolyzed in the newly formed cis ring with a presteady-state burst of ≈6 mol of Pi per mole of 14-mer. No burst is observed when the trans ring contains ADP. The amplitude and kinetics of ATP hydrolysis in the cis ring are independent of the presence or absence of encapsulated substrate protein and independent of K+ at concentrations where there are profound effects on the linear steady-state rate. The hydrolysis of ATP by the cis ring constitutes a second, nonvariable timer of the chaperonin cycle.
Acknowledgments.
We thank Dr. Edward Eisenstein (Center for Advanced Research in Biology, University of Maryland Biotechnology Institute) for the gift of the plasmids pGEL1, pGES1, and pGES1His; Dr. George H. Reed (University of Wisconsin, Madison, WI) for the plasmid pET-E117K PK overexpressing the K+-independent pyruvate kinase mutant E117K; and Dr. Martin Webb (Medical Research Council National Institute for Medical Research, London) for the gift of a plasmid overexpressing the phosphate-binding protein mutant A137C. We also thank Drs. Dorothy Beckett and Dave Thirumalai for constructive criticism. This work was supported by National Institutes of Health Grant 1R01GM06851-01.
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© 2008 by The National Academy of Sciences of the USA. Freely available online through the PNAS open access option.
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Received: April 25, 2008
Published online: November 11, 2008
Published in issue: November 11, 2008
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Acknowledgments
We thank Dr. Edward Eisenstein (Center for Advanced Research in Biology, University of Maryland Biotechnology Institute) for the gift of the plasmids pGEL1, pGES1, and pGES1His; Dr. George H. Reed (University of Wisconsin, Madison, WI) for the plasmid pET-E117K PK overexpressing the K+-independent pyruvate kinase mutant E117K; and Dr. Martin Webb (Medical Research Council National Institute for Medical Research, London) for the gift of a plasmid overexpressing the phosphate-binding protein mutant A137C. We also thank Drs. Dorothy Beckett and Dave Thirumalai for constructive criticism. This work was supported by National Institutes of Health Grant 1R01GM06851-01.
Notes
This article contains supporting information online at www.pnas.org/cgi/content/full/0807429105/DCSupplemental.
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The authors declare no conflict of interest.
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