Some effects of indole on the interaction of amino acids with tryptophanase.
Although indole is a potent inhibitor (KI = 0.01 mM) of pyruvate formation from substrates of tryptophanase (EC 4.1.99.1, from Escherichia coli), we could not detect binding of indole to free tryptophanase (KD greater than 1.0 mM). However, indole, skatole, and toluene increased the affinity of tryptophanase for certain inhibitory amino acids. Binding of amino acids with small side chains (e.g. Ala, Gly) was increased, but there was little or no effect on the binding of amino acids with bulky side chains (e.g. norvaline, ethionine). These effects were quantitated by using changes in the absorption spectra of the enzyme . amino acid complexes. Indole decreases the absorbance obtainable at 500 nm for amino acids with small hydrophobic side chains (L-Ala, Gly), increases this absorbance for amino acids with small polar side chains (beta-cyano-L-alanine), and does not change the spectra of tryptophanase complexes with amino acids with bulky side chains, i.e. amino acids whose binding affinities are unaffected by indole. These spectral differences are interpreted in terms of an effect of bound indole (or side chain binding) on the partitioning of the bound amino acid between catalytic forms of the enzyme. The data indicate that substrate-induced conformational changes occur at the enzyme active site that generate a high affinity indole-binding site during catalytic turnover of tryptophanase and are important in the catalytic functioning of the enzyme. These changes also explain reproducible differences in KI values observed previously for amino acids in different assay systems used for steady state kinetic inhibition studies. The optimal conditions for the growth of E. coli for tryptophanase production are outlined, together with a procedure for purification of holotryptophanase.
D000409:Alanine / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007211:Indoles / Q000494:pharmacology*; D008190:Lyases / Q000378:metabolism*; D012862:Skatole / Q000494:pharmacology; D013053:Spectrophotometry; D013237:Stereoisomerism; D014050:Toluene / Q000494:pharmacology; D014368:Tryptophanase / Q000378:metabolism*
D000596:Amino Acids; D003470:Culture Media; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007211:Indoles; D014050:Toluene; D012862:Skatole; D008190:Lyases; D014368:Tryptophanase; D000409:Alanine