p-Dimethylaminobenzenediazo sodium sulfonate (Fenaminosulf)-reductase from Pseudomonas fragi Bk₉

Abstract

ρ -Dimethylaminobenzenediazo sodium sulfonate (DABDS)-reductase catalyzing the conversion of DABDS to N,N-dimethyl-ρ -phenylenediamine was purified to homogeneity from the cell-free extracts of Pseudomonas fragi Bk₉, crystallized, and its properties studied. The homogeneity of the enzyme was ascertained by gel electrophoresis and immunodiffusion studies. The molecular weight of the enzyme as determined by the gel filtration method was found to be approximately 87,000. The enzyme was optimally active at pH 7 and 45° C, with an activation energy of 2.5 kcal/mol. NADH, NADPH, and GSH could not function as cofactors, while the enzyme required dithiothreitol as an electron donor. From the Lineweaver-Burk plots, the K_m values were calculated to be 0.90 and 3.53 mM for DABDS and dithiothreitol, respectively. The enzyme did not show any requirement for metal ions and was inhibited to varying degrees by different sulfhydryl reagents.