Inactivation of class I fructose diphosphate aldolases by the substrate analog N-bromoacetylethanolamine phosphate.

N-Bromoacetylethanolamine phosphate, prepared by the bromoacetylation of ethanolamine phosphate, has been tested as an active site-specilic reagent for rabbit and rat muscle fructose diphosphate aldolases. The reagent inactivates both enzymes, and inactivation is prevented by substrates or competitive inhibitors. Loss of activity is pseudo-first order until the later stages of inactivation, and a rate-saturation effect is observed as the reagent concentration is increased. The stoichiometry of the reaction has been determined with 14C-labeled reagent. Inactivated enzyme contains 2 to 2.5 molar eq of reagent per mole of aldolase subunit, whereas the enzyme modified in the presence of a competitive inhibitor contains only 1 to 1.5 eq of reagent. Thus, alterations in the catalytic properties of aldolase that accompany its modification apparently reflect the alkylation of a single, essential residue. Characterization of the protein derivatives reveals that the preservation of enzymatic activity is due primarily to the protection of a histidyl residue. This observation supports earlier evidence for the involvement of a histidyl residue in aldolase activity.