Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants.
The 70-kDa heat shock cognate protein is a member of a highly conserved family of molecular chaperones in which the binding and release of target polypeptides are coupled to the chaperones' ATPase activity. The ATPase activity resides in the amino-terminal 44-kDa fragment of the protein. Four acidic residues of the ATPase fragment which might participate in catalysis (Asp-10 and Asp-199, which are Mg2+ ion ligands; Glu-175 and Asp-206, which are candidates for a role as catalytic base) have been individually mutated to both the cognate amide residue (aspartate to asparagine, glutamate to glutamine) and to serine, and the effects of the mutations on the kinetics (this manuscript) and structure (Flaherty, K.M., Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12899-12907) have been determined. Changes at Asp-10 and Asp-199 reduced kcat to approximately 1% of wild type; changes at Glu-175 and Asp-206 reduced kcat to approximately 10% of wild type. Changes to Asp-199 and Asp-206 had little effect on Km; changes to Asp-10 and Glu-175 increased Km 10-100-fold. These data suggest that the bound magnesium ion and its local environment are crucial to catalysis; they argue against a single residue acting as the sole essential general base catalyst in the hydrolytic reaction.
Wilbanks
S M
SM
Beckman Laboratories for Structural Biology, Department of Cell Biology, Stanford University School of Medicine, California 94305.
DeLuca-Flaherty
C
C
McKay
D B
DB
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D018076:DNA, Complementary; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D018076:DNA, Complementary; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; D008274:Magnesium