Adenosine deaminase covalently linked to soluble dextran. The effect of immobilization on thermodynamic and kinetic parameters.
Dextran-linked adenosine deaminase (EC 3.5.4.4) has been prepared. The polymer-linked enzyme possesses an optimal enzymatic activity of 27 units/mg immobilized protein (non-bound enzyme: 200 units/mg protein). Support-bound adenosine deaminase (4.5 microgram protein/mg dextran) shows an enhanced heat stability, a moderately increased Km, and a decreased V value compared to those of the free enzyme. The pH dependences of V and pKm values of dextran-linked adenosine deaminase show only two inflection points compared to three for the free enzyme, which are equivalent to the pK values of the enzyme. Since the missing third inflection point (pH 9.8) can be assigned to the pK value of the epsilon-amino group of lysyl residues, it can be concluded, that immobilization of adenosine deaminase on cyanogen-bromide-activated dextran took place via these lysyl residues. The remaining pK values found from the other inflection points are moderately shifted owing to the altered secondary structure. From the temperature dependence of the enzymatic activity, a 40% decrease of the activation energy of the support-bound enzyme was found, indicating diffusion controlled deamination. The immobilization of adenosine deaminase results in a fluorescence quenching of 80%, without shifting the ultraviolet maximum of the emission spectrum. As already shown for unmodified dextran, the matrix of polymer-linked adenosine deaminase is degradable by a bacterial endodextranase (EC 3.2.1.11).
D000243:Adenosine Deaminase / Q000378:metabolism*; D002850:Chromatography, Gel; D003911:Dextrans / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009700:Nucleoside Deaminases / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D003911:Dextrans; D004800:Enzymes, Immobilized; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase