4-Aminobutyrate aminotransferase fluorescence studies.

The kinetics of resolution of the pyridoxamine phosphate form of the enzyme 4-aminobutyrate aminotransferase were monitored by fluorescence spectroscopy. 2 mol pyridoxamine phosphate are released/mol enzyme, indicating that two molecules of cofactor are involved in catalysis. The apoprotein is reconstituted by addition of pyridoxal phosphate; the apparent rate constant corresponding to the formation of active species is not a linear function of the concentration of cofactor. A multistep mechanism is proposed for the reconstitution of 4-aminobutyrate aminotransferase. A slow phase of reactivation of the aminotransferase is observed when the apoprotein is allowed to reconstitute in the presence of pyridoxal kinase, ATP and pyridoxal. The enzyme 4-aminobutyrate aminotransferase is a dimeric protein made up of subunits of identical molecular weight. It is characterized by a rotational relaxation time of 110 ns. The dimeric structure does not dissociate into subunits over a wide range of protein concentration (4--0.2 micrometer) at neutral pH.