Our investigations of the structure and function of the tryptophan synthase multienzyme complex provide insights into mechanisms of catalysis, metabolite channeling, and allosteric interactions. During the past year, we have investigated the effects of temperature, monovalent cations, and an allosteric ligand on the catalytic properties of tryptophan synthase. Our results provide evidence that increased temperature converts tryptophan synthase from a low activity, open conformation to a high activity, closed conformation under certain conditions. The allosteric ligand and monovalent cations affect the equilibrium between the open and closed forms. Mutations in a regulatory salt bridge between the alpha and beta subunits produce deleterious effects that can be repaired by increased temperature in the presence of certain ligands. To better understand the effects of disease causing mutations in human cystathionine beta-synthase, we have investigated the structure and function of cystathionine beta-synthase from yeast. Our results demonstrate that the N-terminal residues 1-353 compose a catalytic domain and that the C-terminal residues 354-507 compose a regulatory domain. Spectroscopic and kinetic studies establish the overall catalytic mechanism. Comparisons of the yeast and human enzymes reveal significant differences in catalytic and regulatory properties. Importantly, the human enzyme is heme-dependent, whereas the yeast enzyme is heme-independent.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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U.S. National Inst Diabetes/Digst/Kidney
United States
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Jhee, K H; Yoshimura, T; Miles, E W et al. (2000) Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry. J Biochem (Tokyo) 128:679-86
Jhee, K H; McPhie, P; Miles, E W (2000) Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis and regulation. Biochemistry 39:10548-56
Fan, Y X; McPhie, P; Miles, E W (2000) Regulation of tryptophan synthase by temperature, monovalent cations, and an allosteric ligand. Evidence from Arrhenius plots, absorption spectra, and primary kinetic isotope effects. Biochemistry 39:4692-703
Fan, Y X; McPhie, P; Miles, E W (2000) Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge. Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects. J Biol Chem 275:20302-7
Jhee, K H; McPhie, P; Miles, E W (2000) Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein. J Biol Chem 275:11541-4
Miles, E W; Davies, D R (2000) Protein evolution. On the ancestry of barrels. Science 289:1490
Ro, H S; Miles, E W (1999) Structure and function of the tryptophan synthase alpha(2)beta(2) complex. Roles of beta subunit histidine 86. J Biol Chem 274:36439-45
Fan, Y X; McPhie, P; Miles, E W (1999) Guanidine hydrochloride exerts dual effects on the tryptophan synthase alpha 2 beta 2 complex as a cation activator and as a modulator of the active site conformation. Biochemistry 38:7881-90
Miles, E W; Rhee, S; Davies, D R (1999) The molecular basis of substrate channeling. J Biol Chem 274:12193-6