Kinetics of immobilized pig heart fumarase

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Abstract
The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution. An analogous pH dependence of initial activity is found for free and immobilized enzyme. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent Km values are three to eight times higher for the immobilized (2 mg/g gel) enzyme. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ⧎g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration. The activation energies for free and immobilized fumarase (50 ⧎g/g gel) were found to be identical between 22 and 32‡C and with L-malate as substrate (Ea = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases. Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ⧎g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.