The binding of pyridoxamine 5-phosphate to alanine aminotransferase as measured by fluorescence spectroscopy

Abstract

The interaction between pyridoxamine 5-phosphate and alanine aminotransferase ( l-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) was investigated by fluorescence spectroscopy. The cofactor bound to the catalytic site of the enzyme is subject to environmental perturbations which cause a blue shift of 17 mμ in the band position of the emission spectrum, a 10-fold decrease in the fluorescence yield and a uniform increase in the polarization of fluorescence values. An analysis of the results of polarization of fluorescence according to Perrin's equation indicates that pyridoxamine 5-phosphate has rotational flexibility when bound to the catalytic site of the enzyme alanine aminotransferase. Since the fluorescence properties of the cofactor are sensitive to conformational changes of the enzyme, it was possible to investigate the mechanism of inhibition by p-mercuribenzoate and obtain further information on the functional role of the reactive sulfhydryl groups of the enzyme alanine aminotransferase.