Effect of denaturants on the structure and activity of 3-hydroxybenzoate-6-hydroxylase.

The effect of denaturants such as urea, sodium dodecylsulphate (SDS), guanidinium hydrochloride (Gu.HCl) on the structure of enzyme 3-hydroxybenzoate-6-hydroxylase was studied using intrinsic fluorescence and far and near-UV-CD spectroscopic techniques. Also, activity profiles of the enzyme, as a function of increasing concentrations of denaturants were studied. The far-UV CD spectrum of the enzyme did not show appreciable alterations in the presence of urea, SDS or Gu.HCl, thereby suggesting that the protein does not undergo gross conformational changes in its alpha-helical secondary structure. The treatment of enzyme with 2 M urea resulted in almost complete loss of catalytic activity, accompanied by the reduction of emission fluorescence of enzyme. Similarly, treatment with 0.01% SDS also caused almost complete loss of activity and quenching of enzyme fluorescence as well as a red shift in the emission peak. In addition, reduction in the intensity of near-UV-CD spectrum, especially at 280 nm was observed. About 70% of the activity was lost by treatment with 20 mM Gu.HCl, accompanied by quenching of intrinsic fluorescence of the enzyme. The change in intrinsic fluorescence of the enzyme in the presence of 5 mM-100 mM Gu.HCI could be correlated to progressive loss of catalytic activity. Thus, intrinsic fluorescence (due to tryptophan residues) could be used as an effective probe to provide an insight into the relation between the activity and subtle conformational changes of the enzyme. The results suggested that denaturants caused very slight conformational changes in the enzyme that perturbed the microenvironment of aromatic amino acid residues such as tryptophan accompanied by reduction or loss of catalytic activity.

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.