Kinetics of affinity labeling the l-tyrosine/ l-phenylalanine transport system in Bacillus subtilis

Abstract

Kinetic analyses of the irreversible inhibition of l-tyrosine and l-phenylalanine transport in Bacillus subtilis by phenylalanine chloromethyl ketone revealed that the inhibition was due to an affinity labeling process. Phenylalanine chloromethyl ketone is a competetive inhibitor of l-tyrosine and l-phenylalanine transport. The K i values for irreversible inhibition of l-tyrosine and l-phenylalanine transport were 194 and 177 μ m, respectively, and the first order rate constants for the alkylation reaction leading to inactivation of transport of l-tyrosine and l-phenylalanine were 0.016 and 0.012 min −1, respectively. The similarity of these constants are consistent with the involvement of the same functional site for l-phenylalanine and l-tyrosine transport. A second effect of phenylalanine chloromethyl ketone was inhibition of the uptake of neutral, aliphatic amino acids; transport of basic and acidic amino acids was unaffected by it. Since high concentrations of any amino acid did not reduce the inhibitory effects of phenylalanine chloromethyl ketone on transport of neutral, aliphatic amino acids, an independent effect, not due to an affinity labeling process was inferred. A procedure for selective labeling of the l-tyrosine/ l-phenylalanine transport system was demonstrated that should be applicable to the introduction of a radioactive label into the transport protein(s).