Structure And Function Of Tryptophan Synthase And Cystat

Diabetes, Digestive, Kidney Diseases

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Abstract

The tryptophan synthase multienzyme complex from bacteria provides a new paradigm for the investigation of allosteric linkages and the regulation of metabolic channeling. Channeling of an indole intermediate from the tryptophan synthase alpha subunit, where it is formed from indole-3-glycerol phosphate, to the beta subunit, where it reacts with L-serine to form L-tryptophan, is regulated by allosteric interactions transmitted between the alpha and beta sites. Our previous crystallographic and kinetic studies have established that channeling of the indole intermediate through a 25 angstrom long tunnel is regulated by switching of the enzyme between an open conformation of low activity and a closed conformation state of high activity, triggered by ligand binding to the alpha site and covalent transformations at the beta site. During the past year, we have examined the effects of monovalent cation substitution at a site in the beta subunit combined with amino acid substitution of residues that form salt bridges between the alpha and beta subunits. The results show that both cation binding and formation of a salt bridge between alpha subunit Asp56 and beta subunit Lys167 are important to the transmission of allosteric signals between the sites. A second study has established important roles for beta subunit Asp305 both in the conformational change between open and closed states and in substrate binding and recognition. The results this year provide new insights into the relationship between the structure and function of the tryptophan synthase multienzyme complex. Cystathionine beta-synthase catalyzes the synthesis of L-cystathionine from L-serine and L-homocysteine. Mutations in the human enzyme lead to the accumulation of L-homocysteine, which is an important risk factor in coronary heart disease and other vascular diseases. Our previous studies of cystathionine beta-synthase from Saccharomyces cerevisiae established the domain structure and the cofactor dependence of the yeast enzyme. The enzyme is composed of an N-terminal catalytic domain and a C-terminal regulatory domain. The yeast enzyme is solely dependent on the pyridoxal phosphate coenzyme, whereas the human enzyme depends on both pyridoxal phosphate and heme. The spectroscopic properties of the pyridoxal phosphate coenzyme and the absence of heme in the yeast enzyme facilitated our spectroscopic studies that identified several catalytic intermediates and established the overall catalytic mechanism of the enzyme for the first time. During the past year, we have carried out rapid-scanning stopped-flow and single wavelength stopped-flow kinetic measurements under pre-steady-state conditions, as well as circular dichroism and fluorescence spectroscopy under steady-state conditions. The results establish the rates of formation of intermediates in the reaction and demonstrate that the reaction is rate-limited by the conversion of the aminoacrylate intermediate to cystathionine. Further investigation of the substrate specificity of the enzyme has established that L-allo-threonine, but not L-threonine, serves as an effective substrate. The rapid-scanning stopped-flow results show that the 3-methyl-aminoacrylate intermediate, which is observed for the first time, reacts with L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine.