Functional asymmetry in Escherichia coli enzyme I subunits demonstrated by reaction with 3-bromopyruvate

Abstract

In the bacterial phosphoenolpyruvate-dependent sugar transport systems, enzyme I (EI) is responsible for the initial reaction step which is the transfer of the phosphoryl group from phosphoenolpyruvate to a cytoplasmic phosphocarrier protein (HPr). The inactivation of enzyme I by the substrate analogue 3-bromopyruvate has been investigated. Incubation of enzyme I with only micromolar concentrations of this reagent results in complete and irreversible loss of enzymatic activity within a few minutes. Other alkylation reagents such as 2-iodoacetate, 3-bromopropionate, or 5-bromovalerate are far less effective inhibitors of enzyme I, suggesting that the inactivation by 3-bromopyruvate is brought about by the alkylation of one or more essential residues at the active site. Phosphoenolpyruvate and pyruvate, or phosphoenolpyruvate and oxalate, when added together, protect against inactivation by bromopyruvate. Experiments with bromo[2-14C]pyruvate showed that one residue per enzyme I dimer is first alkylated without causing any loss of enzymatic activity. Alkylation of a second residue causes complete inactivation. Both alkylated residues are cysteines. The observations in this report together with published data on the phosphorylation of EI suggest that the subunits of the EI dimer modulate one another's activity during the turnover of the enzyme.