PURIFICATION AND KINETIC-STUDIES ON A PORPHOBILINOGEN DEAMINASE FROM THE UNICELLULAR GREEN-ALGA SCENEDESMUS-OBLIQUUS

Porphobilinogen deaminase, the enzyme condensing four molecules of porphobilinogen, was isolated and purified from light grown Scenedesmus obliquus (wild type). The purification procedure included heat treatment, ammonium sulphate fractionation, gel filtration, high-resolution anion-exchange chromatography and hydrophobic interaction chromatography. The enzyme was purified 1368-fold, compared to the initial crude extract. Its final specific activity was 6812 units.(mg.protein)(-1) at pH 7.4 with a recovery of 44%. The relative molecular mass was 33000, as determined by Sephadex G-100 gel filtration, and 35900 by lithium dodecyl sulfate-polyacrylamide-gel electrophoresis, indicating that the enzyme is a monomer. Studies of initial reaction velocities showed a linear progress curve for hydroxymethylbilane formation and a hyperbolic dependence of the initial reaction rate on substrate concentration, consistent with a sequential displacement mechanism. Apparent kinetic constants (K-m and V-max) for the conversion of porphobilinogen to hydroxymethylbilane at 37 degrees C, pH 7.4, were 79 mu M and 176 pmol.min(-1), respectively. Variation of both V-max and K-m with pH indicated the presence of ionizable groups in the enzyme-substrate complex(es), showing a single ionization (pK 7.15) in V-max/K-m plots. A sharp pH-profile for V-max was interpreted as a positive cooperative proton dissociation. In spite of the two pathways existing for 5-aminolevulinate biosynthesis in Scenedesmus, currently there is no indication of the existence of two porphobilinogen deaminases or even of isoenzymes.