Tryptophan synthase alpha subunit glutamic acid 49 is essential for activity. Studies with 19 mutants at position 49.

Yutani, Katsuhide; Ogasahara, Kyoko; Tsujita, Tadahiro; Kanemoto, K; Matsumoto, Mitsuharu; Tanaka, Shun‐ichi; Miyashita, Takanori; Matsushiro, Aizo; Sugino, Yoshinobu; Miles, Edith Wilson

doi:10.1016/s0021-9258(19)76444-8

Cited by 68 publications

(15 citation statements)

References 34 publications

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“…Role of Glu Residue at Position 49 in the Conformational Stability of the Wild-Type a-Subunit. Nineteen mutant proteins, with substitutions at position 49 of the -subunit, have been prepared, and the enzymatic activities of all these mutant proteins have been examined (Yutani et al, 1987a). All of them lost their catalytic activity in the process of the formation of Trp from indole-3-glycerol phosphate, indicating that the Glu residue at position 49 of the -subunit plays a crucial role in the catalysis.…”

Section: Discussionmentioning

confidence: 99%

“…The trpA33 strain (Yanofsky & Horn, 1972) was donated by Dr. C. Yanofsky. The mutant gene of the Thr92 strain was obtained through site-directed mutagenesis using an oligodeoxynucleotide (Yutani et al, 1987a). The trpA33 strain has Met in place of Glu at position 49 of the -subunit, and the Thr92 strain has Thr in place of His at position 92.…”

Section: Methodsmentioning

confidence: 99%

“…For preparation of the -subunit with phenyW5-alanine substituted at all 12 Phe residues, E. coli strain KMBL1788 (thyA30¡, bio87, argA, endAlOl, pheA, metE72) was used as a host for the pUCSPtrptrpA containing the trpA gene (Yutani et al, 1987a) because of its requirement of Phe. The broth used was M9-glucose, to which approximately 0.04 mg/mL each of 19 amino acids other than Phe, 0.1 mg/mL thymine, 0.05 mg/mL biotin, and 0.04 mg/mL phenylalanine (MSD Isotopes) were added.…”

Section: Methodsmentioning

confidence: 99%

“…of the correlation between the structure of a protein and its stability or specific function. Mutant proteins derived from the tryptophan synthase -subunit of Escherichia coli have been studied to elucidate the roles of its amino acid residues in protein folding and protein stability (Yutani et al, 1977(Yutani et al, , 1979(Yutani et al, ,1980b(Yutani et al, , 1982(Yutani et al, , 1984(Yutani et al, , 1985Ogasahara et al, 1984Ogasahara et al, , 1985 or to elucidate the mechanism of its function (Hodo et al, 1977; Patterson et al, 1977;Yutani et al, 1987a). Our previous results indicated that single amino acid substitutions at a unique position have a marked effect on the conformational stability of a protein, depending on the nature of the substituted residues (Yutani et al, 1984).…”

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confidence: 99%

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Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase .alpha.-subunits

Akutsu¹,

Ogasahara²,

Tsujita³

et al. 1987

Biochemistry

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In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs. Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92. The replacement did not affect the conformation of the protein significantly. The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues. On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH. NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another. The present results are discussed in the light of the conformational stability of the protein.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase .alpha.-subunits

Akutsu¹,

Ogasahara²,

Tsujita³

et al. 1987

Biochemistry

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“…The determination of the X-ray crystal structure has been instrumental in the identification of those amino acid residues which are likely either to play important roles in catalysis or in the allosteric function of the < •ß2 bienzyme complex (Hyde et al, 1988; Hyde & Miles, 1990). Use of this information to select -subunit residues for site-directed mutagenesis has resulted in the identification of catalytic residues within the -active site (Nagata et al, 1989; Miles et al, 1988; Yutani et al, 1987). Furthermore, mutation of certain a-subunit residues has been shown to alter the accumulation and dis-tribution of intermediates covalently bound at the ß active site (Kawasaki et al, 1987;Brzovic'et al, 1992).…”

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confidence: 99%

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the .beta.-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium

Brzović¹,

Kayastha²,

Miles³

et al. 1992

Biochemistry

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In an effort to understand the catalytic mechanism of the tryptophan synthase beta-subunit from Salmonella typhimurium, possible functional active site residues have been identified (on the basis of the 3-D crystal structure of the bienzyme complex) and targeted for analysis utilizing site-directed mutagenesis. The chromophoric properties of the pyridoxal 5'-phosphate cofactor provide a particularly convenient and sensitive spectral probe to directly investigate changes in catalytic events which occur upon modification of the beta-subunit. Substitution of Asp for Glu 109 in the beta-subunit was found to alter both the catalytic activity and the substrate specificity of the beta-reaction. Steady-state kinetic data reveal that the beta-reaction catalyzed by the beta E109D alpha 2 beta 2 mutant enzyme complex is reduced 27-fold compared to the wild-type enzyme. Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy shows that the mutation does not seriously affect the pre-steady-state reaction of the beta E109D mutant with L-serine to form the alpha-aminoacrylate intermediate, E(A-A). Binding of the alpha-subunit specific ligand, alpha-glycerol phosphate (GP) to the alpha 2 beta 2 complex exerts the same allosteric effects on the beta-subunit as observed with the wild-type enzyme. However, the pre-steady-state spectral changes for the reaction of indole with E(A-A) show that the formation of the L-tryptophan quinonoid, E(Q3), is drastically altered. Discrimination against E(Q3) formation is also observed for the binding of L-tryptophan to the mutant alpha 2 beta 2 complex in the reverse reaction. In contrast, substitution of Asp for Glu 109 increases the apparent affinity of the beta E109D alpha-aminoacrylate complex for the indole analogue indoline and results in the increased rate of synthesis of the amino acid product dihydroiso-L-tryptophan. Thus, the mutation affects the covalent bond forming addition reactions and the nucleophile specificity of the beta-reaction catalyzed by the bienzyme complex.

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Structural Basis for Catalysis by Tryptophan Synthase

Miles

1991

Advances in Enzymology - And Related Areas of Molecular Biology

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Tryptophan synthase alpha subunit glutamic acid 49 is essential for activity. Studies with 19 mutants at position 49.

Cited by 68 publications

References 34 publications

Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase .alpha.-subunits

Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase .alpha.-subunits

Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the .beta.-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium

Structural Basis for Catalysis by Tryptophan Synthase

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