[Reaction of fluorescein bimercuric acetate with a molybdenum center of xanthine oxidase from milk].
Inhibition of milk xanthine oxidase by fluorescein bimercuriacetate (FMA) allows for the classification of S-containing groups according to their localization and role in the catalytic activity of the enzyme. The enzyme (E) complexes with FMA (E--FMA I and E--FMA II) differing in their activity, stoichiometry and spectral properties were studied at various experimental conditions, reaction time and FMA concentrations. The enzyme molecule contains 5 groups that are reactive towards FMA (E--FMA I) and are localized outside the active center. That these groups have no concern with activity and are subjected to modification irrespective of whether or not the xanthine oxidase molecule has an intact Mo-center. The formation of an inactive E--FMA II complex is associated with an additional (in comparison with E--FMA I) binding of two FMA molecules per molecule of the active enzyme. The stoichiometry of the E--FMA II complex was determined by the X-ray fluorescent method from the amount of the Hg in enzyme. A kinetic scheme of xanthine oxidase inhibition by FMA is proposed, according to which the inhibition is a result of modification of two groups in the enzyme active center, of which only one is essential for the enzyme activity. This scheme also postulates the role of reversible E--FMA complexes in the course of irreversible inhibition. Xanthine oxidase is protected against FMA by the substrate (xanthine), competitive inhibitors (azaxanthine and allopurinol) and acceptor (2,6-dichlorophenolindophenol), i. e., compounds which interact with the Mo-center of the enzyme. The EPR spectra of the dithionite-reduced E--FMA II complex were found to contain a "slow" signal, Mo(V), typical of the Mo-center devoid of labile sulphur. It was assumed that the essential group interacting with FMA in the active center of xanthine oxidase as a terminal sulphur which is a component of the coordination region of Mo.
Kozachenko
A I
AI
Nagler
L G
LG
Lependina
O L
OL
Ianovskaia
I M
IM
Vartanian
L S
LS
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D005452:Fluoresceins; D007202:Indicators and Reagents; D007700:Kinetics; D008892:Milk / Q000201:enzymology*; D008982:Molybdenum / Q000378:metabolism*; D009941:Organomercury Compounds; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors*
D005452:Fluoresceins; D007202:Indicators and Reagents; D009941:Organomercury Compounds; D013438:Sulfhydryl Compounds; C001673:fluorescein mercuric acetate; D008982:Molybdenum; D014969:Xanthine Oxidase
Reaktsiia fluorestseinbimerkuriatsetata s molibdenovym tsentrom ksantinoksidazy iz moloka.