Cytosolic aspartate aminotransferase: Studies on subunit interactions with the apo/holo hybrid dimer

Abstract

The apo/holo hybrid dimer of cytosolic aspartate aminotransferase from pig heart was compared with the apo/apo and holo/holo homomer with respect to manifestations of subunit interactions. The hybrid dimer was isolated by iso-electric focusing from an apo enzyme preparation semi-reconstituted with pyridoxal-5′- P. The binomial distribution pattern of pyridoxal/pyridoxal homomer, apo/pyridoxal hybrid, and apo/apo homomer obtained by the iso-electric separation as well as identical rates of binding of the coenzyme to the apo/holo hybrid and the apo/apo homomer indicate random association of the coenzyme with the apo subunits. An extensive comparative exploration of the active site regions of the apo/holo hybrid containing one active site and the holo/holo homomer containing two revealed no differences with respect to the following structural and functional properties: catalytic center activity, absorption and circular dichroism spectra of the coenzyme chromophore, p K′ a values of enzyme-bound pyridoxal-5′- P, K′ m and V′ values for aspartate and 2-ketoglutarate, the dissociation constants of the complex with competitive dicarboxylic inhibitors, the absorption spectrum and the rate of decay of the intermediate with erythro-3-hydroxyaspartate. The rate of alkylation of Cys-390 with N-ethylmaleimide was found to be about 20 times faster in the apo/apo homomer than in the pyridoxal/pyridoxal homomer. The rates of modification of the apo and of the pyridoxal subunit in the hybrid are the same as those in the respective homomers. Likewise, the syncatalytic conformational changes of the holo subunit do not affect the reactivity of Cys-390 on the adjoining apo subunit. Thus, by manifold criteria, the conformational changes that accompany transamination do not reflect subunit interactions but rather are connected with the catalytic processes at functionally isolated active sites.