Studies on chlorophyllase. The mechanism of the action of lecithin liposomes on enzyme activity and the function of the carbohydrate moiety of the enzyme

A sensitive and continuous fluorescence assay of chlorophyllide formation in the presence of lecithin liposomes has been developed. The mechanism of the enhancing effect of lecithin on chlorophyllase-catalyzed hydrolysis of chlorophyll has been elucidated. Using both fluorescence and biochemical assays, the function of the concanavalin A-reactive carbohydrate moiety of chlorophyllase has been investigated. Experiments on the interaction of chlorophyllase with concanavalin A show that the sugar group not only stabilizes the enzyme, but also is essential for the manifestation of enzyme activity. From a comparison of the results obtained with solubilized and membrane-bound enzyme, it is concluded that the active site is situated on the outside of the thylakoid membrane. Mg²⁺, in combination with dithiothreitol, activates chlorophyllase. In the presence of Mg²⁺, an abnormal pH dependence of the inhibiting effect of concanavalin A on chlorophyllase-catalyzed chlorophyll hydrolysis and the reversibility of this effect through the addition of α-methyl-D-mannoside have been observed. The cause of this atypical reaction as well as of other Mg²⁺ effects is discussed.