Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

The photoinactivation of the Δ (5)-3-ketosteroid isomerase of Pseudomonas testosteroni in the presence of 3-oxo-4-estren-17β-yl acetate and air is accompanied by destruction of histidine and aspartate (or asparagine). The first order rate constant of photoinactivation of the enzyme is equal, within experimental error, to the first order rate constant for the destruction of a single aspartate (or asparagine) residue and is considerably greater than the first order rate constant for the destruction of a single residue of histidine. When the photolysis is carried out under anaerobic conditions, only aspartic acid (or asparagine) is destroyed as enzyme is inactivated. Both inactivation and aspartate (or asparagine) destruction occur to a greater extent in the absence of oxygen than in its presence. The destruction of histidine, on the other hand, is found to be strictly oxygen-dependent. These results suggest that photochemical modification of a single residue of aspartate (or asparagine) is largely, if not entirely, responsible for photoinactivation of the enzyme under these conditions. When irradiated in the presence of 3-oxo-4-entren-17β-yl acetate, performic acid-oxidized bovine pancreatic ribonuclease does not suffer any detectable destruction of its aspartic or asparaginyl residues but does undergo significant destruction of its histidine residues. These observations suggest that the aspartate (or asparagine) residue modification found with isomerase is an active site-directed photochemical reaction, whereas the modification of histidine may not be.