~5.4 refolded luciferase/DnaK
||Bacteria Escherichia coli
||Sharma SK, De los Rios P, Christen P, Lustig A, Goloubinoff P. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat Chem Biol. 2010 Dec6(12):914-20. doi: 10.1038/nchembio.455. p.916 right column bottom paragraphPubMed ID20953191
||P.914 right column 2nd paragraph: "Here [investigators] clarified the mechanistic subprocesses of the Escherichia coli version of the Hsp70-Hsp40-NEF chaperone system, the DnaK-DnaJ-GrpE system, by using a preformed and well-characterized misfolded substrate. [They] denatured a firefly luciferase mutant by a freeze-thaw treatment and isolated a near-homogenous population of stable, misfolded compact inactive monomers. The unique qualities of this chaperone substrate allowed for the first time the measuring of the rates of the various steps leading from a stable inactive misfolded state to a stable, native, enzymatically active refolded state. This permitted [them] to correlate rates of ATP consumption with rates of refolding to the native state. The data showed that bacterial Hsp70, DnaK, uses the energy of ATP to convert misfolded substrates into unfolded intermediates that, upon release from the chaperone, can spontaneously refold to their native state."
||P.916 right column bottom paragraph: "DnaK ATPase and refolding at substrate saturation: As mentioned, direct measurement of the maximal rate Vmax of chaperone-assisted refolding activity has not been possible for any of the ATPase chaperones. The determination of the molecular activity of any chaperone (mol substrate turned over to product per mol chaperone and unit time) has thus remained out of experimental reach. With [investigators’] new experimental system in hand, [they] proceeded to measure classical enzyme kinetics, using DnaK, DnaJ and GrpE at constant low concentrations (0.4 μM, 0.3 μM, 0.8 μM, respectively) in the presence of ATP and varying substrate concentrations. [They] observed the formation of native luciferase at yields that increased linearly during the first minutes of the reaction (Fig. 4a), confirming that disaggregation is not rate limiting, which is at variance with previously used assays (refs 17, 18, 27). Additionally, initial rates increased with the substrate concentration, following saturation kinetics with an apparent Vmax′ value of about 5.4 refolded luciferase molecules per DnaK molecule and a Km′ value of 2 μM, which corresponds to a fivefold molar excess of substrate over enzyme (Fig. 4b). Remarkably, excess substrate did not inhibit the refolding reaction, as observed with previously used DnaK substrates, such as small soluble heat-generated aggregates of glucose-6-phosphate dehydrogenase (ref 18) or with other chaperones and substrates, such as GroEL-GroES and urea-denatured RubisCO (ref 7)."