Fig. 5. CupB5 peptides do not compete with MucB for MucA binding but stimulate WVF-activated AlgW cleavage of MucA.

(A) Release of ³⁵S-MucB from MucA-agarose following incubation with buffer, MucA (155 μM), L-IIA (20 mg/mL), or CupB5 peptides (~300 μM). For the CupB5 peptides, values are averages ± SEM (n=3). For L-IIA, the value is the average ± SEM (n=2). (B) Rates of cleavage by AlgW (0.5 μM trimer) and WVF peptide (150 μM) were measured at different concentrations of ³⁵S-MucA in the presence or absence of CupB5 peptides (300 μM). Values are averages ± SEM (n=2) and were fit to the Hill form of the Michaelis-Menten equation, rate = V_max/(1 + (K_M/[substrate])^h). (C) Rates of cleavage of ³⁵S-MucA (50 μM) by AlgW (0.5 μM trimer) were assayed at difference concentrations of WVF peptide in the presence or absence of CupB5 peptides (300 μM). Values are averages ± SEM (n=2) and were fit to the equation rate = basal + V_max/(1 + (K_act/[peptide])^h). Without CupB5 peptide, h was 2.2 ± 1, K_act was 11 ± 2 μM, and V_max was 1250 ± 150 M⁻¹ s⁻¹. With GYYYTVV peptide, h was 1.5 ± 0.8, K_act was 18 ± 7 μM, and V_max was 2490 ± 490 M⁻¹ s⁻¹.