Abstract
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 mutant proteins have been determined. All three mutant proteins are devoid of measurable ATP hydrolysis activity. Crystal structures of the mutant proteins have been determined to a resolution of 1.7 A; 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.
Highlights
The bovine 70-kDa heat shock protein (Hsc70)1 is thought to “chaperone” protein folding by, at a minimum, binding to nascent or misfolded segments of polypeptides, thereby inhibiting their aggregation
The results suggested a pathway for ATP hydrolysis in which MgATP would first bind with the Mg2ϩ ion complexed through outer sphere coordination to the carboxyl groups of Asp-10 and Asp-199 and with a nonbonded oxygen of the -phosphate of ATP bound directly to the ion [1]
The fact that we have observed no activity whatsoever with these proteins combined with the fact that we have previously made reliable measurements of ATPase activities with other mutant proteins whose turnover rates were 100-fold slower than wild-type protein argues strongly that all three Lys-71 mutants are devoid of ATPase activity
Summary
The bovine 70-kDa heat shock protein (Hsc70) is thought to “chaperone” protein folding by, at a minimum, binding to nascent or misfolded segments of polypeptides, thereby inhibiting their aggregation (for reviews, see Refs. 3 and 4). The ATPase domain can be isolated as an independent, fully functional entity either as a proteolytic fragment from full-length protein [10, 12] or as a recombinant expression product [2, 13]. This allows the option of studying the ATPase. Mechanism on this domain alone, thereby circumventing the complexities encountered in the full-length protein where the ATPase activity is coupled to peptide binding and release. Such a reaction scheme proposes that Lys-71 would be a catalytically essential residue
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