Kinetics and Mechanism of the Recognition of Endotoxin by Polymyxin B

We report here, for the first time, the elementary steps involved in the recognition of the endotoxin (lipopolysaccharide) molecule by polymyxin B (PMB), a cyclic cationic decapeptide. Miniscule amounts of lipopolysaccharide, the invariant structural component of Gram-negative bacterial outer membranes, in circulation in humans elicits "endotoxic shock" syndrome, which is fatal in almost 60% of the instances. PMB, despite its harmful side effects, is the only useful drug for combating endotoxic shock. It neutralizes the endotoxin by binding to it. The kinetics and mechanism of this important biological recognition, investigated by stopped-flow spectrofluorometry, provide considerable insight about the endotoxin neutralizing activity of this antibiotic. This process consists of a pair of kinetically distinguishable but consecutive association and dissociation reactions, with rate constants of 1.98 × 105 M-1 s-1 (k1), 0.458 s-1 (k2), 0.458 s-1 (k-1), and 0.0571 s-1 (k-2) at 20 °C. Analysis of the activation parameters for this recognition suggests that, during the first phase of the reaction, which is bimolecular in nature, PMB associates with endotoxin in a relatively less constrained manner. Subsequently, a considerable reorganization of this initial complex occurs, which entails a significant expenditure of energy. Design of analogues of PMB which are able to overcome the constraints of the rate-limiting step should serve as more effective agents for treating endotoxic shock.