Some structural aspects of vanadium bromoperoxidase from Ascophyllum nodosum.
The stability of the vanadium containing bromoperoxidase from Ascophyllum nodosum was studied. The enzyme was very resistant against chemical denaturation. Denaturation did not occur upon incubation in 4 M guanidine hydrochloride. Circular dichroism measurements showed that the secondary structure was not affected upon incubation in 4% sodium dodecyl sulphate. The sedimentation coefficient and the molecular mass, determined by ultracentrifugation were 6.96 S and 97 kDa, respectively, indicating a very compact molecule. The protein molecule contained 16 cysteine residues, all of which participated in the formation of disulfide bridges. Circular dichroism-measurements in the far ultraviolet region revealed that the protein consisted of a large amount of alpha-helix (74%), and no beta-pleated sheet. The dissociation constant of the apoprotein vanadium-complex was 55 nM (at pH 8.5), and rapidly increased at lower pH. The data suggest that the protonation of a group with a pKa higher than 8.5 prevents the binding of vanadate. Structural analogues of vanadate (phosphate and arsenate) were competitive inhibitors with respect to the reconstitution of the bromoperoxidase. The inhibition constants were 60 and 120 microM for phosphate and arsenate, respectively. The binding of hydrogen peroxide to the enzyme was visualized by optical spectroscopy. Upon addition of H2O2 the optical absorption spectrum showed a small, but significant, decrease in absorption in the 315 nm region, which was restored upon addition of bromide, or by allowing the solution to stand for several hours. These changes are ascribed to the formation of a stable enzyme-peroxo-intermediate, in line with a previous analysis of the steady-state kinetics.
Tromp
M G
MG
E.C. Slater Institute for Biochemical Research and Biotechnological Centre, University of Amsterdam, The Netherlands.
Olafsson
G
G
Krenn
B E
BE
Wever
R
R
D002942:Circular Dichroism; D004795:Enzyme Stability; D056890:Eukaryota / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D010544:Peroxidases; C026195:bromide peroxidase; D019791:Guanidine