Lysine 71 of the chaperone protein Hsc70 is essential for ATP hydrolysis

It has been proposed that lysine 71 of the bovine 70-kDa heat shock cognate protein might participate in catalysis of ATP hydrolysis by stabilizing an H2O molecule or an OH- ion for nucleophilic attack on the gamma-phosphate of the nucleotide (Flaherty, K, M., Wilbanks, S, M,, DeLuca-Flaherty, C., and McKay, D. B, (1994) J, Biol. Chem, 12899-12907; Wilbanks, S, M,, DeLuca-Flaherty, C,, and McKay, D, B. (1994) J. Biol. Chem, 269, 12893-12898), To test this hypothesis, lysine 71 of the ATPase fragment 70-kDa heat shock cognate protein has been mutated to glutamic acid, methionine, and alanine; and the kinetic and structural properties of the mutantproteins have been determined. All three mutant proteins are devoid of measurable ATP hydrolysis activity. Crystal structures of the mutant proteins have bben determined to a resolution of 1.7 Angstrom; all three have ATP in the nucleotide binding site. These data identify lysine 71 as a residue that is essential for chemical hydrolysis of ATP.