On the microenvironment of the pyridoxamine 5-phosphate residue in NaBH 4 -reduced glycogen phosphorylase b. Absorption and fluorescence studies.

Abstract

The absorption and fluorescence properties of NaBH4‐reduced glycogen phosphorylase b were studied and used for probing the microenvironment of the pyridoxamine 5′‐phosphate residue and for monitoring structural transitions occurring in the enzyme. Free pyridoxamine 5′‐phosphate in a solvent mixture composed of about 80% dimethylformamide and 20% water was found to simulate the optical properties of the enzyme at neutral pH (absorption maxima, molar absorption, fluorescence excitation and emission maxima and quantum yield). Therefore it is suggested that at pH 7, where the enzyme exhibits maximal catalytic activity, the pyridoxamine 5′‐phosphate residue is “buried” in a hydrophobic microenvironment. Upon gradually lowering the pH of the medium to 4.8, there are structural changes in the enzyme which are reflected in an increase of the molar absorption around 330 nm and of the quantum yield of the cofactor fluorescence. These transitions can be simulated with free pyridoxamine 5′‐phosphate by increasing the water content of the dimethylformamide‐water mixture to 80–90%. It seems, therefore, that acidification of the medium causes structural changes in the enzyme which expose the cofactor site.