Open AccessPurification and properties of NADH/NADPH‐dependent p ‐hydroxybenzoate hydroxylase from Corynebacterium cyclohexanicum
Author(s) -
FUJII Takaaki,
KANEDA Toshi
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb08724.x
Subject(s) - hydroxybenzoate , enzyme , chemistry , molecular mass , biochemistry , chromatography , polyacrylamide gel electrophoresis , enzyme assay , cofactor , stereochemistry , acrylamide , monomer , organic chemistry , polymer
Crude soluble extracts of Corynebacterium cyclohexanicum , grown on cyclohexanecarboxylic acid, were found to contain 4‐hydroxybenzoate 3‐hydroxylase which functions with NADH as well as NADPH. The purified enzyme preparation was electrophoretically homogeneous and contained FAD as prosthetic group. The relative molecular mass of the enzyme was estimated to be about 47000 by native and denaturated acrylamide gel electrophoresis, indicating that it is monomeric. The enzyme was stable at 60°C for 10 min. The enzyme was highly specific for p ‐hydroxybenzoate. The activity was inhibited by several aromatic analogues of p ‐hydroxybenzoate such as p ‐aminobenzoate, p ‐fluorobenzoate, o ‐hydroxybenzoate, m ‐hydroxybenzoate, 2,4‐dihydroxygenzoate, and 2,5‐dihydroxybenzoate. The K m value for NADH was fairly constant, about 45 μM, in the pH range 7.0 – 8.4, whereas the K m value for NADPH increased from 63 μM to 170 μM as the pH rose from 7.0 to 8.4. V values in the same pH range, however, were approximately constant in both cases; about 30 μmol min −1 mg −1 for NADH, and 26 μmol min −1 mg −1 for NADPH, and 26 μmol min −1 mg −1 for NADPH. Mg 2+ was required for full activity of the enzyme in low concentrations of phosphate buffer. The enzyme was inhibited by Cl − which was non‐competitive with respect to NADH, NADPH and p ‐hydroxybenzoate.