4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It converts the neurotransmitter 4-aminobutyric acid to succinic semialdehyde. In order to study the structural and functional aspects of catalytically active Cys residues of pig brain 4-aminobutyrate aminotransferase, we purified the aMive form in E. coli by coproduction of thioredoxin. The structural arrangement for functional requirements of a dimeric protein using a bifunctional sulfhydryl reagent was then characterized, and the spatial proximity between the essential SH groups and a cofactor (pyridoxal-5`-phosphate) binding site was determined. The bifunctional sulfhydryl reagent DMDS reacted with the enzyme at the ratio of one molecule per enzyme dieter. This resulted in an approximately 50 % loss of enzymatic activity. The spatial proximity of the distance between the essential SH groups and the cofactor-binding site was determined by the energy transfer measurement technique. The result (approximate 20 A) suggested that cross-linking of two sulthydryl groups with DMDS is not near a PLP binding site.