Induction of vancomycin resistance in the Gram-positive Enterococci requires a two-component regulatory system, VanS and VanR, for transcriptional activation of three genes (vanH, A, X) that encode enzymes for a cell wall biosynthetic pathway that produces an altered peptidoglycan intermediate with lower affinity for the antibiotic. The catalytic efficiency (kcat/KM) has been determined for phosphotransfer from the phosphohistidyl form of VanS to both its homologous partner VanR and the heterologous (Escherichia coli) response regulator PhoB. The rate of formation of the phosphoaspartyl forms of VanR and PhoB were determined as well as the rate of appearance of inorganic phosphate. Using PhoB in excess of P-VanS, a pseudo-first-order rate constant (kxfer) of 0.2 min-1 for phosphotransfer and a KM for PhoB of 100 μM were readily determined. The corresponding kxfer of 96 min-1 for phosphotransfer from P-VanS to VanR required rapid quench kinetics. A KM of 3 μM was estimated for VanR, leading to a 104-fold p...