ATPase kinetics of recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal ATPase domain.
Steady-state kinetic, pre-steady-state kinetic, and equilibrium binding measurements have been applied to determine the rate constants of individual steps of the ATPase cycle for the recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal 44 kDa ATPase fragment. At 25 degrees C, pH 7.0, in the presence of 75 mM KCl and 4.5 mM Mg2+, the measured association rate constants for MgATP approximately hsc70 and MgADP approximately hsc70 are (2.7 +/- 0.5) x 10(5) and (4.1 +/- 0.5) x 10(5) M-1 s-1, respectively, while the dissociation rate constants are 0.0114 (+/- 0.0002) and 0.0288 (+/- 0.0018) s-1, respectively. MgATP (Kd = 0.042 microM) therefore binds to hsc70 more tightly than MgADP (Kd = 0.11 microM). ADP release is inhibited by inorganic phosphate (Pi), suggesting that product dissociation is ordered with Pi released first and ADP second. The rate of chemical hydrolysis of ATP is 0.0030 (+/- 0.0003) s-1 for hsc70 and 0.0135 (+/- 0.0033) s-1 for the 44 kDa fragment. The rate of Pi release is 0.0038 (+/- 0.0010) s-1 for hsc70 and 0.0051 (+/- 0.0006) s-1 for the 44 kDa fragment. For the 44 kDa fragment, Pi release is the slowest step in the ATPase cycle, while for hsc70, Pi release and chemical hydrolysis of MgATP have similar rates; in both cases, ADP release is a relatively rapid step in the ATPase cycle.
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J H
JH
Beckman Laboratories for Structural Biology, Department of Structural Biology, Stanford University School of Medicine, California 94305.
McKay
D B
DB
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D010710:Phosphates; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases