To shed light on the mechanism of hydrophobic control in reactions of microbial tryptophanase the direct effect of the solvent hydrophobicity on affinities of amino acid inhibitors was first examined. Values of inhibition constants (K-i) for a variety of amino acids were determined in 37.5% aqueous methanol, and no general correlation between the change of K-i, on passing from water to aqueous methanol, and amino acid hydrophobicity was found. The solvent effects on the separate stages of the external aldimine formation (K-D) and deprotonation to form a quinonoid intermediate (K-q) were determined for the reactions of tryptophanase with 2-oxindolyl-L-alanine and L-alanine by stopped-flow technique. For 2-oxindolyl-L-alanine, which is a close transition-state analogue for the enzyme reaction with natural substrate, the decrease in the affinity in aqueous methanol is associated exclusively with the alpha-proton abstraction stage but not with the preceding formation of external aldimine. We conclude that the environment of amino acid side chains in the active site cannot be considered to be permanently hydrophobic irrespective of the bound amino acid. We suggest that complexes of tryptophanase with amino acids may exist either in a hydrophobic, presumably "closed", conformation, where bound amino acids are isolated from the solvent, or in an accesible to solvent, "open", conformation, depending on the structure of the bound amino acid and stage of the catalytic mechanism. For 2-oxindolyl-L-alanine the transfer from an open to a closed conformation probably accompanies deprotonation of the external aldimine. The change of the active site hydrophobicity may provide an efficient way of modulating the relative acid-base properties of the catalytic groups to ensure the movement of protons in the "correct" direction depending on the elementary stage of catalysis. (C) 2003 Elsevier Science Inc. All rights reserved.