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The synthesis of L-tryptophan from indole and L-serine, as catalyzed by the B subunit of Escherichia coli tryptophan synthetase, has been studied with steady-state and rapid-reaction kinetic techniques. Initial velocity measurements of the reaction have been made utilizing the absorption difference between indole and tryptophan at 289 nm. The results were consistent with both a compulsory sequence of substrate addition and with a random, rapid equilibration between enzyme and substrates. Dissociation constants and/or Michaelis constants for serine and indole were obtained. Temperature-jump, stopped-flow, and combined stopped-flow-temperature-jump measurements made on solu-

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E. coli NADPH-sulfite reductase, depleted of FMN but retaining its FAD, has been prepared by photoirradiation of native enzyme in 30% — saturated ammonium sulfate. FMN-depleted enzyme loses its ability to reduce (using NADPH) ferricyanide, cytochrome c, sulfite, or the enzyme’s own heme-like chromophore. However, the FAD remains rapidly reducible by NADPH, and the FMN-depleted enzyme retains NADPH-acetylpyridine NADP* transhydrogenase activity. Thus, FAD can serve as entry port for NADPH electrons, and FMN is required for further transmission along the enzyme’s electron transport chain. These data, plus other studies, have enabled us to suggest a mechanism for catalysis which involves FAD cycling between the fully-oxidized and fully-reduced forms while FMN cycles between fully-reduced and semiquinone. This mechanism, which includes a disproportionation step, permits a “step-down” from the twoelectron donor, NADPH, to a succession of equipotential one-electron transfer steps.

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