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Thermodynamic analysis of purified human placental α-l-fucosidase

par Hoebeke, Johan;Groswasser, José ;Bousard, Nelly;Vamos, Eszter ;Strosberg, Arthur
Référence Archives of biochemistry and biophysics, 198, 2, page (556-561)
Publication Publié, 1979-12

Article révisé par les pairs

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Résumé :

The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pKm were for the 4-methylumbelliferyl-conjugate ΔF = -6.6 kcal/mol, ΔH = -8.5 kcal/mol, and ΔS = -6.3 e.u. and for the p-nitrophenylconjugate ΔF = -5.6 kcal/mol, ΔH = -12.2 kcal/mol, and ΔS = -21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = -12.4 kcal/mol and ΔS = -20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the "aglycone" moiety of the fluorogenic substrate to the enzyme. © 1979.