Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis.
The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.
O'Brien
M C
MC
Beckman Laboratories for Structural Biology, Department of Cell Biology, Stanford University School of Medicine, California 94305.
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; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction
D002352:Carrier Proteins; D018076:DNA, Complementary; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D013912:Threonine; D000255:Adenosine Triphosphate