Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein).
Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1 +/- 0.1 microM and 1.4 +/- 0.7 mM respectively at 25 degrees C. This corresponds to delta G0' = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, delta H0' was calculated to -36 +/- 2 kJ/mol. This gives delta S0' = 6.7 J/mol per K. Adenosine 5'-[gamma-thio]triphosphate, ADP, adenosine 5'-[beta, gamma-imino]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 microM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.
Buxbaum
E
E
School of Biological Science, University of Manchester, U.K.
Woodman
P G
PG
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D007887:Fabaceae / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D066298:In Vitro Techniques; D007700:Kinetics; D010946:Plants, Medicinal; D013552:Swine; D013816:Thermodynamics
D002352:Carrier Proteins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases