As mentioned above, phospholipases are distinguished from general esterases by the fact that they interact with interfaces in order to function. The difference in reaction velocity with substrate concentration for these two types of enzymes is illustrated in Fig. 7.9. Whereas esterases show classical Michaelis-Menten kinetics, the phospho-lipases show a sudden increase in activity as the substrate (phospholipid) concentration reaches the critical micellar concentration (CMC) and the molecules tend to form aggregates or micelles with polar ends in the aqueous environment (Fig. 7.10). Because of the physical nature of the substrate, enzyme activity is dependent both on the (hydro-
phobic) interaction with the aggregate and on the formation of a catalytic Michaelis complex. Several factors have been suggested as being responsible for the increased rate of hydrolysis at interfaces:
(1) the very high local substrate concentration;
(2) substrate orientation and physical state at the interface;
(3) increased rates of diffusion of products from the enzyme;
(4) increased enzyme activity due to conformational changes on binding to the interface.
For a typical phospholipid in solution, formation of a micelle increases its effective concentration by at least three orders of magnitude - depending on the CMC (factor 1 above). The British biochemist Dawson and also Dutch workers including van Deenen, de Haas and Slotboom have studied how the nature of aggregated lipid (factor 2) influences markedly the activity of phospholipases. Such activity seems to depend on four parameters -surface charge, the molecular packing within the aggregate, the polymorphism of the aggregate and the fluidity of the phospholipid's acyl chains. The surface charge can be quite different from the bulk pH and is influenced by ionic amphipaths as well as ions in the aqueous environment. This explains why, for example, the phospholipase B from Penicillium notatum will not attack pure phosphati-dylcholine but is active in the presence of activators, such as phosphatidylinositol, which give phosphatidylcholine micelles a net negative charge. Molecular packing is particularly important for the acylhydrolases, but is also relevant to the phos-phodiesterases (phospholipase C). Changes in activity with this parameter have been referred to in the example of diethylether activation above. Polymorphic states can include micelles, bilayer structures and hexagonal arrays (Section 6.5.3). There is some evidence that certain phospholipases attack preferentially hexagonal structures and since these are believed to be formed transiently at sites of membrane fusion (Section 6.5.10), the phospho-lipase may help to remove fusative lipid and help re-establish the normal membrane bilayer. In addition, it has been well-established that gel-phase lipids are attacked preferentially by phospholipases from several sources.
Factor 3 is particularly important for phospholi-pases A and B where both products from a typical membrane (natural) phospholipid are hydrophobic. In the case of phospholipases C and D, where one product is water-soluble, action at an interface is still useful because the water-soluble product can move into the aqueous environment while the other product can diffuse into the hydrophobic phase. Similar physico-chemical arguments explain why many phospholipases show increased activity in the presence of organic solvents. Thus, when diethylether is used with phospholipase A assays it is thought that accumulating fatty acids are more readily removed. In addition, the solvent may allow more ready access of the enzyme to the hydrocarbon chains of the phospholipid and, by reducing micellar size, increases the effective surface area for reaction. Finally, conformational changes (factor 4) have been shown to take place when some digestive phospholipases interact with substrate and/or Ca2+. Kinetic studies indicate that such a conformation change is needed for maximal activity.
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