Their K 1/2 values scaled inversely with the aggregate lengths of their connecting segment between Tsr1 and Tsr2: the longest linker (546~551/L-6) produced the most sensitive response (K 1/2 = 55 μM); the shortest (517~551/L-4) failed to respond to serine, but evinced high kinase activity upon potassium cyanide challenge, which depletes cellular ATP, the phosphodonor for the CheA autophosphorylation reaction (see Methods ).