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.
Sumathi
S
S
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012. sumathis@iitb.ac.in
Dasgupta
Dipak
D
D001419:Bacteria / Q000201:enzymology; D002942:Circular Dichroism; D019791:Guanidine / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
D012967:Sodium Dodecyl Sulfate; D014508:Urea; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase; D019791:Guanidine