The tryptophan synthase α2β2 complex: a model for substrate channeling, allosteric communication, and pyridoxal phosphate catalysis.

I reflect on my research on pyridoxal phosphate (PLP) enzymes over fifty-five years and on how I combined research with marriage and family. My Ph.D.

Effects of hydrostatic pressure on the conformational equilibrium of tryptophan synthase from Salmonella typhimurium.

A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase.

Pressure and temperature jump relaxation kinetics of the conformational change in Salmonella typhimurium tryptophan synthase L-serine complex: large activation compressibility and heat capacity changes demonstrate the contribution of solvation.

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase.

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase alpha2beta2 multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium alpha-glycerophosphate (Na2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of betaE109D and betaD305A mutations, on K(eq) for the conformational equilibria were determined.

Hydrostatic pressure affects the conformational equilibrium of Salmonella typhimurium tryptophan synthase.

The effect of hydrostatic pressure on the tryptophan (Trp) synthase alpha2beta2 complex from Salmonella typhimurium has been investigated. Trp synthase has been shown previously to exhibit low-activity (open) and high-activity (closed) conformations. The equilibrium between the open and closed conformations of Trp synthase has been found to be affected by a wide range of variables, including alpha-subunit ligands, monovalent cations, organic solvents, pH, and temperature.

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole.

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate.

Detection of open and closed conformations of tryptophan synthase by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance of bound 1-15N-L-tryptophan.

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site.

Allosteric communication in the tryptophan synthase bienzyme complex: roles of the beta-subunit aspartate 305-arginine 141 salt bridge.

The allosteric interactions that regulate substrate channeling and catalysis in the tryptophan synthase bienzyme complex from Salmonella typhimurium are triggered by covalent reactions at the beta-site and binding of substrate/product to the alpha-site. The transmission of these allosteric signals between the alpha- and beta-catalytic sites is modulated by an ensemble of weak bonding interactions consisting of salt bridges, hydrogen bonds, and van der Waals contacts that switch the subunits between open and closed conformations. Previous work has identified a scaffolding of salt-bridges extending between the alpha- and beta-sites consisting of alphaAsp 56, betaLys 167, and betaAsp 305.

Yeast cystathionine beta-synthase reacts with L-allothreonine, a non-natural substrate, and L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine.

Our studies of the reaction mechanism of cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of L-serine with L-homocysteine to form L-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions.