Possible Site-Specific Reagent for the General Amino Acid Transport System of Saccharomyces cerevisiae

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids. The amino acid analogue N-5-chloroacetyl-L-ornithine (NCAO) has been tested as a potential site specific reagent for this system. l-Tryptophan, which is transported exclusively by the general transport system, was used as a substrate. In the presence of glucose as an energy source, NCAO inhibited tryptophan transport competitively (Ki=80 μM) during short time intervals (1-2 min), but adding 100 μM NCAO to a yeast cell suspension resulted in a time-dependent activation of tryptophan transport during the first 15 min of treatment. Following the activation a time-dependent decay of tryptophan transport activity occurred. Approximately 80% inactivation of the system was observed after 90 min. When a yeast cell suspension was treated with NCAO in the absence of an energy source, an 80% inactivation of tryptophan transport occurred in 90 min. The inactivation was noncompetitive (Ki ⋍ μM) and could not be reversed by the removal of the NCAO. Addition of a fivefold excess of L-lysine during NCAO treatment or prevented inactivation of tryptophan transport. Under parallel conditions of incubation, other closely related transport systems were not inhibited by NCAO.