THREONINE-204 OF THE CHAPERONE PROTEIN HSC70 INFLUENCES THE STRUCTUREOF THE ACTIVE-SITE, BUT IS NOT ESSENTIAL FOR ATP HYDROLYSIS

The chaperone protein Hsc70 is an ATPase of unknown mechanism, althoug h the crystal structure of the 44-kDa ATPase domain has been solved. T his structure shows that the hydroxyl of threonine 204 is located clos e to the gamma-phosphate of ATP, in a position where it might be an in termediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) a nd threonine to glutamic acid (T204E). The wild-type ATPase domain had a K(m) for ATP of approximately 1 muM; the mutants had K(m) values of approximately 90 muM. The k(cat) 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-angs trom resolution, respectively. The overall tertiary structure of the m utants showed little change from the wild type; however, significant c hanges were observed in the active site. Analysis of the structures su ggested possible reasons for the changes in kinetic constants. Threoni ne 204 does not seem to be an obligatory intermediate phosphate accept or in the hydrolysis reaction since the mutants retained appreciable A TPase activity.