Fig. 4

Functional characterisation of the amino acid binding site. a Substrate specificity of GkApcT analysed using counterflow of the 20 canonical amino acids plus d-Ala and a water control (emp) using external ³H l-Ala. n = 3 independent experiments, error bars s.d. b View showing the molecular volume of the side chains within the binding site of the WT GkApcT structure. Due to the size of Met321, there is no space to accommodate the extra side chain bulk of l-Arg. c Effect of mutating residues in the binding site to their counterparts in CAT-2A. Accumulation was measured using ³H l-Ala uptake using counterflow accumulation with internal l-Ala, l-Arg or l-lys. n = 3 independent experiments, error bars s.d. d Crystal structure of the Met321Ser variant with bound Arginine (purple), shown in sticks. The mFo-DFc difference electron density map contoured at 3 σ, (green mesh). e Representative IC₅₀ curves determined for l-Arg binding to either the WT or M321S variant of GkApcT using an electrogenic uptake assay. IC₅₀ values were calculated from three independent replicates