Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH

Abstract

4‐Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time‐dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, фobs= 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL≈ 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1‐anilinonaphthalene‐8‐sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped‐flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped‐flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4‐aminobutyrate aminotransferase in the cell. Copyright © 1995, Wiley Blackwell. All rights reserved