Similarities and differences in interactions of the activity-enhancing chemoreceptor pentapeptide with the two enzymes of adaptational modification.

Sensory adaptation and chemotaxis by Escherichia coli require a specific pentapeptide at the chemoreceptor carboxyl terminus. This sequence binds the two enzymes of receptor adaptational modification, enhancing catalysis, but with different binding features and mechanisms. We investigated the relative importance of each pentapeptide side chain for the two enhancing interactions.

Dihydroquinone ansamycins: toward resolving the conflict between low in vitro affinity and high cellular potency of geldanamycin derivatives.

Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.

Allosteric enhancement of adaptational demethylation by a carboxyl-terminal sequence on chemoreceptors.

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme.