ATP HYDROLYSIS IS NOT REQUIRED FOR THE DISSOCIATION OF A SUBSTANCE P-CENTER-DOT-BIP COMPLEX

BiP is a member of the hsp70 family of proteins that is present in the endoplasmic reticulum where it functions as a molecular chaperone. Ra pid quantitative assays have been used to study the effect of mutating BiP residue 229, located in the ATP binding site, from threonine to g lycine. Although binding of ATP to the mutant BiP was not affected, th e mutant protein possessed 10-20% of the wild-type BiP ATPase activity . Binding to a model peptide substrate, substance P (Brot ef al. (1994 ) Proc. Natl, Acad. Sci. USA 91, 12120-12124), was twofold higher with mutant BiP at 4 degrees C than with wild-type BiP, and was ATP depend ent. Under these conditions the substance P that was bound to mutant B iP, but not the wild-type, could be released by higher levels of ATP ( 5-10 mu M),and the ratio of substance P released to ATP hydrolyzed was greater than 10, These results suggest that stoichiometric ATP hydrol ysis is not required for release of a chaperone from its substrate. (C ) 1996 Academic Press, Inc.