Inactivation of sortase A mediated by metal ATCUN complexes

Catalytic metallopeptides that target the membrane-associated sortase A transpeptidase have been developed and evaluated as irreversible inactivators of SrtA∆N59 (sortase A, lacking the initial membrane-binding domain). The copper-binding GGH tripeptide ATCUN motif was linked to amidated forms of the cell wall sorting signal, LPET and LPETG, as sortase-targeting moieties. The resulting metallopeptides were used to determine half maximal inhibitory concentrations (IC₅₀) and rate constants for time-dependent sortase A inactivation. Michaelis-Menten behavior was observed for the catalytic metallopeptides, and k(cat), K(M) and k(cat)/K(M) parameters were obtained as 0.080 ± 0.002 min⁻¹, 23 ± 2 μM and 0.0035 ± 0.0003 μM⁻¹ min⁻¹, respectively. Concentration-dependent inhibition of SrtA∆N59 by the metallopeptides revealed IC₅₀ values ranging from 570 to 700 µM, while Cu-GGH, which lacked a targeting motif, had no measurable IC₅₀ value (>2,000 µM). Time-dependent inactivation of SrtA revealed a range of catalytic activities, with Cu-GGHGLPETG-NH2 demonstrating the fastest rate of inactivation in the presence of ascorbate and hydrogen peroxide coreactants. The active site of the enzyme comprises residues Cys-184, Arg-197 and His-120. LC-MS/MS analysis of the reaction products demonstrated modification of Cys-184 to cysteine sulfonic acid (+48 amu). Results obtained from a DTNB assay support oxidation of the Cys-184 residue. LC-MS/MS also suggested oxidation of the Arg-197 containing peptide. 2D NMR analysis was performed to assess the possible oxidation of His-120, however, none was observed. These compounds possess the potential for irreversible inactivation of SrtA through oxidative modification of essential residues required for substrate binding.