Chemical modification of 3-HBA-6-hydroxylase by phenylglyoxal: kinetic and physicochemical studies on the modified enzyme.

The inactivation of 3-HBA-6-hydroxylase isolated from Micrococcus species by phenylglyoxal and protection offered by 3-HBA against inactivation indicate the presence of arginine residue at or near the substrate binding site. The loss of enzyme activity was time and concentration dependent and displayed pseudo-first order kinetics. A 'n' value of 0.9 was obtained thus suggesting the modification of a single arginine residue per active site which led to the loss of enzyme activity. The enzyme activity could be restored by extensive dialysis at neutral pH. Quenching of the intrinsic fluorescence and reduction in the ellipticity value at 280 nm in the near-UV CD spectrum of the enzyme was noticed after its treatment with phenylglyoxal. These observations probably imply distinct perturbations in the environment of adjacent aromatic amino acid residues such as tryptophan as a consequence of arginine modification.