Front Matter

This model has been derived from kinetic data of the action of cobra venom PLA 2 in

mixed micelles with the nonionic surfactant Triton X-100.

Both models yield mathematically equivalent kinetics, and are appropriate to fit

the experimental results. On special substrates forming large anionic vesicles, Jain

and Berg (1989) analyzed whether PLA 2 remains associated with the interface while

hydrolyzing successive phospholipid substrate molecules (‘scooting mode’) or

whether it dissociates from the interface after each catalytic event (‘hopping

mode’). They found a preference for the scooting mode with this substrate.

The phenomenon of interfacial activation can also be observed with other phos-

pholipases such as PLC (Hough and Hansen, 1994) and PLD (Allgyer and Wells,

1979; Nakagaki and Yamamoto, 1984; Lambrecht et al., 1992) where it has

been, however, less thoroughly analyzed hitherto. As studied on the catalysis of

PLC or PLD on monolayers, surface pressure (Quarles and Dawson, 1969; Good-

man et al., 1996) and charge (Chen and Barton, 1971; Kondo et al., 1994b) of the

monolayer strongly influence the activation effect.

Another interesting feature in the action of many phospholipases that is related to

the phenomenon of interfacial activation is the lag-burst behavior in the initial phase

of the catalyzed reaction. In difference to most enzymes with soluble substrates, the

initial rates are not the maximum rates. In the case of phospholipases, maximum rates

are reached only after a latency period. Studies of this phenomenon on PLA 2 (Cal-

lisen and Talmon, 1998), PLC (Basa ́n ̃ez et al., 1996; Ruiz-Argu ̈ello et al., 1998), and

PLD (Dorovska-Taran et al., 1996) supported the hypothesis that full enzyme activity

requires some kind of membrane surface irregularities or defects, originating from

the products of the initial molecular turnovers. Therefore, phospholipase activity

appears to be controlled by the overall geometry and composition of the phospho-

lipid aggregates.

12.4.3 Consequences of kinetic particularities to characterization

of phospholipases

Since phospholipases preferentially hydrolyze substrates in aggregated form, their

activities depend not only on the substrate and enzyme concentrations but also on the

physical state of the substrate, i.e., on the organization and dynamics of the inter-

phase where catalysis occurs. One of the most important prerequisites for reasonable

kinetic measurements is the reproducible preparation of substrate aggregates. Pre-

ferably uniform aggregates such as monolayers, micelles formed from short-chain

phospholipids, liposomes, or mixed micelles formed from phospholipids and addi-

tional surfactants are used. Classical kinetic concepts are not valid for phospholi-

pases, although kinetic analyses by the Michaelis – Menten approach or similar

models are often used. These yield apparent parameters which can be applied

only for the comparison of relative affinities and maximum velocities of different

enzymes under exactly the same conditions of substrate preparation. Therefore, com-

parisons of results obtained in different laboratories are of restricted value, and most

findings on substrate specificities cannot be generalized. Furthermore, the structure

of the substrate aggregates will change during the reaction due to the arising products

236 12 Phospholipases Used in Lipid Transformations