Aromatic amino acid transport in Yersinia pestis

The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-Menten-like kinetics with apparent Km values of 6 x 10(-7) to 7.5 x 10(-7) and 2 x 10(-6) M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10(-6) M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 TO8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact alpha amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and tyrosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhydryl reagents or sodium azide. The results on the selective inhibition of aromatic amino acid transport provide additional evidence for multiple transport systems . These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy.