4‐Aminobutyrate Aminotransferase Fluorescence Studies

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Abstract
The kinetics of resolution of the pyridoxamine phosphate form of [pig brain] 4-aminobutyrate aminotransferase (EC 2.6.1.19) were monitored by fluorescence spectroscopy. Two moles pyridoxamine phosphate are released/mol enzyme, indicating that 2 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 multi-step 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 is a dimeric protein made up of subunits of identical MW. 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 .mu.M) at neutral pH.