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 co gnate protein might participate in catalysis of ATP hydrolysis by stab ilizing an H2O molecule or an OH- ion for nucleophilic attack on the g amma-phosphate of the nucleotide (Flaherty, K, M., Wilbanks, S, M,, De Luca-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 A TPase fragment 70-kDa heat shock cognate protein has been mutated to g lutamic acid, methionine, and alanine; and the kinetic and structural properties of the mutantproteins have been determined. All three mutan t proteins are devoid of measurable ATP hydrolysis activity. Crystal s tructures of the mutant proteins have bben determined to a resolution of 1.7 Angstrom; all three have ATP in the nucleotide binding site. Th ese data identify lysine 71 as a residue that is essential for chemica l hydrolysis of ATP.