title: Reversible dissociation/association of D-amino acid transaminase subunits: properties of isolated active dimers and inactive monomers.
abstract: 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.
authors.affiliation: Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
authors.forename: K
authors.initials: K
authors.lastname: Kishimoto
authors.forename: C
authors.initials: C
authors.lastname: Yasuda
authors.forename: J M
authors.initials: JM
authors.lastname: Manning
mesh_terms: D002850:Chromatography, Gel; D002942:Circular Dichroism; D051263:D-Alanine Transaminase; D019281:Dimerization; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol; D009154:Mutation; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008623:Mercaptoethanol; D000637:Transaminases; D051263:D-Alanine Transaminase