Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH

Pineda, Teresa; Osei, Yaa Difie; Churchich, Jorge E.

doi:10.1111/j.1432-1033.1995.0683m.x

Cited by 4 publications

(4 citation statements)

References 21 publications

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“…The monomeric form of the malate dehydrogenase enzyme present at pH 5.0 was recently shown by hydrogen exchange to be folded into a conformation similar to that adopted by the native dimer at pH 7.0 [25]. The dimeric enzyme 4-aminobutyrate aminotransferase also dissociates into monomers at pH 5.0 [26]. The monomers have similar secondary structure to the dimer.…”

Section: Comparison Of Stabilities At Ph 40 and 70mentioning

confidence: 98%

Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)

Bajaj

Chakshusmathi

Bachhawat-Sikder

et al. 2004

Biochemical Journal

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The protein CcdB (controller of cell division or death B) is an F-plasmid-encoded toxin that acts as an inhibitor of Escherichia coli DNA gyrase. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size-exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The stability of both forms was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal x mol(-1) (1 cal approximately 4.184 J) and 21 kcal x mol(-1) at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (C(m)) of the dimer is dependent on protein concentration, whereas the C(m) of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdmCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdmCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of Delta C (p) (representing the change in excess heat capacity upon protein denaturation) is 2.8+/-0.2 kcal x mol(-1) x K(-1) for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration.

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Section: Comparison Of Stabilities At Ph 40 and 70mentioning

confidence: 98%

Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)

Bajaj

Chakshusmathi

Bachhawat-Sikder

et al. 2004

Biochemical Journal

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“…Rowlett et al (13) measured the dissociation constants between the two different types of subunits (R and β) of tryptophan synthase by mixing different ratios of the separate types of subunits and then measuring the amount of activity generated by the subunit complex. 4-Aminobutyrate transaminase also undergoes a reversible process of dissociation/ association that is pH-dependent but occurs at rates much faster than for D-amino acid transaminase (26). At low pH (pH 5.0) the rate of dissociation of dimers to monomers was reported to be 56 s -1 for 4-aminobutyrate transaminase compared to 7.8 h -1 for D-amino acid transaminase (Figure 6b).…”

Section: Discussionmentioning

confidence: 95%

“…At low pH (pH 5.0) the rate of dissociation of dimers to monomers was reported to be 56 s -1 for 4-aminobutyrate transaminase compared to 7.8 h -1 for D-amino acid transaminase (Figure 6b). However, the association of monomers to form active dimers at pH 7.0 for 4-aminobutyrate transaminase was too fast to be detected even for stopped-flow instruments (26).…”

Section: Discussionmentioning

confidence: 99%

Reversible Dissociation/Association of d-Amino Acid Transaminase Subunits: Properties of Isolated Active Dimers and Inactive Monomers

1999

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The crystal structure of dimeric D-amino acid transaminase shows that the two Trp-139 sites are located in a hydrophobic pocket at the interface between the subunits and that the two indole side chains face one another and are within 10 A of coenzyme. This enzyme prefers an aromatic character at position 139, as previously demonstrated by the finding that Phe-139 but no other substitution tested provides the maximum degree of thermostability and catalytic efficiency. Here we show that an equilibrium between active dimers and inactive monomers can be demonstrated with the W139F mutant enzyme, whereas with the wild-type enzyme the subunit interface is so tight that a study of this equilibrium is precluded. We show how the processes of dimerization of monomers and dissociation of dimers to monomers are controlled. Lower pH (5.0) favors monomer formation from dimers. Gel filtration and activity analysis show that at higher pH (7.0) the monomers combine to form active dimers with a K(d) of 0.17 microM. This assembly process is relatively slow and takes several hours for completion, thereby permitting accurate measurement of kinetics and equilibrium parameters. Absorption and circular dichroism spectra of dimers and monomers are significantly different, indicating that the environment around the cofactor is very likely altered between them. The circular dichroism peak of the W139F dimer at 418 nm is less negative than that of the wild-type enzyme in accordance with its lower visible absorbance; the circular dichroism peak of the W139F monomer at 418 nm is more negative than that of the wild-type enzyme. The dissociation of dimers to monomers has also been studied by taking advantage of these spectral differences, thus permitting the rates of the dissociation and the reassociation to be calculated and compared. 2-Mercaptoethanol assists in the conversion of monomers to dimers. The results here describe dissociation/reassociation in the dimeric enzyme under native conditions without denaturants.

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“…The monomeric form of the malate dehydrogenase enzyme present at pH 5 was shown by hydrogen exchange to be folded into a conformation similar to that adopted by the native dimer at pH 7 [33]. The dimeric enzyme 4-aminobutyrate aminotransferase also dissociates into monomers at pH 5 [34]. The monomers have similar secondary structure to the dimer.…”

Section: Discussionmentioning

confidence: 98%

Thermal, Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

Biswas

Chattopadhyaya

2014

PLoS ONE

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Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol−1 and 14.90 Kcal mol−1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp) is 3.42 Kcal mol−1 K−1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.

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Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH

Cited by 4 publications

References 21 publications

Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)

Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)

Reversible Dissociation/Association of d-Amino Acid Transaminase Subunits: Properties of Isolated Active Dimers and Inactive Monomers

Thermal, Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

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