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K Yutani

Publications and source records attributed to K Yutani.

At least 109 records · Page 6Linked to original sources
PubMed1987

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

We have obtained a complete set of 20 variants of the alpha subunit of tryptophan synthase of Escherichia coli at position 49 in order to extend our previous studies on the effects of single amino acid replacements at position 49 on structure and function. Thirteen mutant alpha subunits have been newly constructed by site-directed mutagenesis using oligonucleotides. Six mutants were available from previous studies. We find that the wild type and all of the mutant alpha subunits form alpha 2 beta 2 complexes with the beta 2 subunit of tryptophan synthase with similar association constants and similarly stimulate the activity of the beta 2 subunit in the synthesis of L-tryptophan from L-serine and indole. Thus none of the changes at position 49 produces a change in the conformation of the alpha subunit which significantly interferes with normal subunit interaction. However, the 19 mutant alpha 2 beta 2 complexes are completely devoid of activity in reactions normally catalyzed by the active site of the alpha subunit. This is the first time that these several activities have been measured with a series of highly purified alpha subunits altered by mutation at a single site. Our finding that the mutant in which glutamic acid 49 is substituted by aspartic acid is totally devoid of alpha activity is especially significant and is strong evidence that glutamic acid 49 is an essential catalytic base in the reaction catalyzed by the alpha subunit. This result is consistent with the results of previous genetic studies, with evolutionary comparisons using sequence analysis, and with recent results from x-ray crystallography of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium.

Base Sequence↗
PubMed1987

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

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.

Amino Acids↗
PubMed1987

Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase alpha subunit.

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, we have constructed a series of variant alpha subunits of tryptophan synthase from Escherichia coli substituted by each of 20 amino acids at position 49, which is buried in the interior of the protein. The stabilities were quantitatively examined except for the mutant protein substituted by arginine, which was not obtained in enough quantity. The Gibbs energy of unfolding in water and the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride for each protein were compared at pH 7.0 and pH 9.0. The Gibbs energy of unfolding in water at pH 7.0 varied from 0.72 to 1.92 times that of the wild-type protein by the substitutions, but the activation Gibbs energy of unfolding in 3 M guanidine hydrochloride varied only from 0.95 to 1.03 times that of the wild-type protein. Moreover, the stability of the protein substituted at this position, which is buried in the interior of the molecule, tended to increase linearly with increasing hydrophobicity of the substituted residue, unless the volume of the substituted residue was over a certain limit.

Amino Acid Sequence↗
PubMed1985

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit.

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.

Glutamates↗
PubMed1985

Effect of amino acid substitutions on conformational stability of a protein.

This paper reviews studies on thermostable proteins from thermophilic bacteria and on mutant proteins of human hemoglobin, tryptophan synthase alpha-subunit of E. coli, T4 phage lysozyme, and phage lambda repressor with respect to the role of the constituting amino acid residues in stabilization of conformation. The stability of a protein is easily affected by single amino acid substitutions, by which the protein undergoes change(s) of one or more of the following: a hydrogen bond, a salt bridge, a hydrophobic interaction, the volume of the residue, a disulfide bond, or the relative position of two aromatic rings.

Amino Acid Sequence↗
PubMed1984

Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase alpha-subunit.

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized.

Amino Acids↗
PubMed1984

Comparison of denaturation of tryptophan synthase alpha-subunits from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid.

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase alpha subunit have been compared by circular dichroism measurements. The three alpha subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (alpha-2 domain) and the N-terminal domain comes from S. typhimurium (alpha-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 degrees C showed that the alpha-2 domain of S. typhimurium was more stable than the alpha-2 domain of E. coli, but the alpha-1 domain of S. typhimurium was less stable than the alpha-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 degrees C and coincided with that obtained from guanidine hydrochloride-induced denaturation.

Crosses, Genetic↗
PubMed1984

Thermal denaturation of tryptophan synthase alpha-subunit. Comparison of the values of thermodynamic parameters of unfolding obtained from van't Hoff analysis of CD measurement with those from calorimetry.

Thermal denaturation for the wild-type of tryptophan synthase alpha-subunits from E. coli and one of its mutant proteins was followed by CD measurements at various pHs in the alkaline region and the results from van't Hoff analyses of the thermal denaturation curves were compared with those from calorimetry. Although the far-u.v. CD spectra of the thermally denatured proteins differed from those of the completely denatured states in 3.2 M guanidine hydrochloride, the titration curves by denaturants at higher temperatures were not sigmoidal but straight lines, indicating that the cooperative structure of the proteins has been completely destroyed by heating. The ratio of calorimetric enthalpy change to van't Hoff enthalpy change obtained from calorimetric study was unity, indicating that the thermal denaturation of the proteins was a two-state system. The unfolding heat capacity change (delta Cp) of the wild-type protein from van't Hoff analysis of the thermal denaturation curves by CD measurement was estimated to be 2.45 kcal/mol X deg, which was similar to that from calorimetry. The values of unfolding enthalpy change at denaturation temperatures were lower by about 15 kcal/mol compared to those from calorimetry.

Calorimetry↗
PubMed1982

Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains.

The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated. Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unfolded form of the alpha subunit at intermediate concentrations of either denaturant. The possibility that this partially unfolded form of the alpha subunit results from the preferential unfolding of one of the two domains of the alpha subunit is now investigated. This study utilizes two proteolytic fragments of the alpha subunit, alpha-1 and alpha-2, which have been shown to refold independently and to correspond to two domains of the alpha subunit. The effects of guanidine hydrochloride concentration on the separate alpha-1 and alpha-2 fragments, on the intact alpha subunit, and on the derivative nicked by trypsin (alpha') are compared by measuring ellipticity at 222 nm and by measuring the susceptibility of tyrosyl residues to chemical modification. The results show that guanidine hydrochloride induced unfolding of the alpha subunit results from the stepwise unfolding of the two domains: the alpha-2 fragment and the corresponding domain in the intact alpha subunit are unfolded by low concentrations of guanidine hydrochloride; the alpha-1 fragment and the corresponding domain in the intact alpha subunit are unfolded by higher concentrations of guanidine hydrochloride.

Dose-Response Relationship, Drug↗
PubMed1982

Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins.

Heat-denaturation of tryptophan synthase alpha-subunit from E. coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins. The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller. Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively. Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees. As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins. The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.

Calorimetry, Differential Scanning↗
PubMed1980

State of Tyr49 in a mutant tryptophan synthase alpha-subunit substituted at position 49.

The states of tyrosine residues in an alpha-subunit of wild-type tryptophan synthase from Escherichia coli and a mutant protein which has tyrosine in place of glutamic acid at position 49, were examined by absorption spectrum, spectrophotometric titration of phenolic hydroxyl ionization, and deuteration kinetics of phenolic hydrogen monitored by fluorescence measurement. The difference absorption spectrum of the mutant protein against the wild-type protein at pH 7.0 and 25 degrees C had peaks at 289, 282, and 276.5 nm. These positions corresponded to those in the absorption spectrum of L-tyrosine derivatives in a non-aqueous solvent at 77 K and these bands were well-resolved even at 25 degrees C as if the tyrosine residue were fixed at lower temperature. The titration curve of the mutant protein at 3 degrees C differed from that of the wild-type protein only above pH 12.7, where the difference molar extinction coefficient at 295 nm reached a plateau, indicating that ionization of Tyr49 took place at an abnormally high pH. These results suggest that Tyr49 is buried in the hydrophobic interior and fixed in a certain orientation. The deuterium exchanges of phenolic hydrogen at pH 7.0 and 13 degrees C in the wild-type and mutant proteins consisted of a single and two first order processes, respectively, all three having smaller rate constants than that of free tyrosine, indicating that these tyrosine residues are buried. It is concluded that Tyr49 in the mutant protein is not on the surface of the molecule.

Chemical Phenomena↗
PubMed1979

Comparison of denaturation by guanidine hydrochloride of the wild type tryptophan synthase alpha-subunit of Escherichia coli and two mutant protein (Glu 49 replaced by Met or Gln).

In order to elucidate the roles of individual amino acid residues in the conformational stability of proteins, the denaturation by guanidine hydrochloride of the wild-type trytophan synthase alpha-subunit of Escherichia coli and two mutant proteins, trpA33 (Glu 49 leads to Met) and trpA11 (Glu 49 leads to Gln), has been compared by means of CD measurements at pH 7.0 and various temperatures. CD spectra of the two mutant proteins were similar to that of the wild-type protein. The trpA33 and the trpA11 proteins were more and less resistant, respectively, to guanidine hydrochloride than the wild-type protein at 9.7 to 49.6 degrees C. The free energy change of unfolding in water delta delta Gnd H2O, was evaluated assuming a three state denaturation, since the denaturation curves of three proteins suggested the presence of one stable intermediate. The values of delta Gnd H2O for the trpA33, the wild-type, and the trpA11 proteins at 25.8 degrees C and pH 7.0 were 13.4,8.8, and 6.3 kcal/mol, respectively. The delta Gnd H2O of the trpA11 protein was almost independent of temperature, though that of the trpA33 protein was remarkably dependent on temperature. The conformation stabilities of the three proteins were correlated with the hydrophobicities of the substituted amino acid residues.

Circular Dichroism↗