Binding of Pyridoxal 5-Phosphate to Cystathionase

Abstract

Abstract The binding of pyridoxal 5-phosphate to the apoprotein of the enzyme cystathionase from rat liver was investigated by two independent methods, absorption and fluorescence spectroscopy. The increase in absorbance at 525 nm associated with Schiff's base formation was used to investigate the binding of pyridoxal-5-P at a protein concentration of 1 x 10-5 m. A model based on two classes of independent binding sites fits the spectrophotometric data reasonably well. The affinity constant determined for the class of weak binding sites (K2 = 1.4 x 104 m-1) corresponds to extraneous binding of pyridoxal-5-P to lysine residues which are not implicated in catalysis. In order to avoid interference by extraneous binding of the cofactor to the enzyme, binding studies were conducted at a protein concentration of 2 x 10-6 m using the method of protein fluorescence quenching. This method gives a value of Ka = 7 x 105 m-1 for the class of tight binding sites. It is proposed that the catalytic subunits of the enzyme cystathionase display similar affinity constants for the cofactor pyridoxal-5-P.