Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase.
No major structural alteration of alkaline phosphatase can be observed in the early stages of enzyme oxidative inactivation by the ascorbate model system. Fluorescence changes of protein-bound 8-anilino-1-naphthalenesulfonic acid suggest, however, that localized modifications take place. Oxidized alkaline phosphatase displays less catalytic efficiency (decrease of Vmax), while retaining the other kinetic properties, including the same affinity for substrates and inhibitors and the same activation energy of the native enzyme. Typical features of the modified protein are a decreased thermal stability and a biphasic heat inactivation profile, which make the oxidized form quite similar to aged enzymes. The lower response to Mg2+ activation indicates that the magnesium binding sites of alkaline phosphatase are probably the targets of the ascorbate system oxidative modifications.
Mordente
A
A
Istituto di Chimica Biologica, Università Cattolica del S. Cuore, Facoltà di Medicina e Chirurgia Agostino Gemelli, Roma, Italy.
Martorana
G E
GE
Miggiano
G A
GA
Meucci
E
E
Santini
S A
SA
Castelli
A
A
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D006899:Mixed Function Oxygenases; D000469:Alkaline Phosphatase; D008274:Magnesium