Inactivation of Rhodanese by Pyridoxal 5′‐Phosphate

Abstract

Pyridoxal 5′‐phosphate and other aromatic aldehydes inactivate rhodanese. This inactivation reaches higher extents if the enzyme is in the sulfur‐free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5′‐phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5′‐phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate–rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5′‐phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship between loss of activity and pyridoxal 5′‐phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5′‐phosphate.