MECHANISM OF BINDING OF SUBSTRATE-ANALOGS TO TRYPTOPHAN INDOLE-LYASE - STUDIES USING RAPID-SCANNING AND SINGLE-WAVELENGTH STOPPED-FLOW SPECTROPHOTOMETRY

We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1 -methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques. Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations. The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm. This species is formed more rapidly than with the other analogues (84.5 s−1) and is reprotonated very slowly (0.2 s−1). Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s−1). The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine. Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed. The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at <4 s−1. These results demonstrate that the structure of the aromatic rings of amino acids can have significant effects on both the rates of formation and the equilibrium concentrations of intermediates in the reaction of tryptophan indole-lyase and suggest that the indole NH is important for optimal substrate binding and catalysis.© 1990, American Chemical Society. All rights reserved.