Front Matter

lipids can also undergo thermotropic phase changes reflecting the fluidity of the fatty

acid chains.

While in vivo phospholipases attack phospholipid clusters of biological mem-

branes, phospholipid substrates in vitro are prepared as liposomes or micelles, often

under addition of synthetic surfactants promoting the solubilization of the phospho-

lipids and thus providing uniform aggregate structures.

12.4.2 Interfacial activation of phospholipases

As is generally valid for lipolytic enzymes, phospholipases are more active towards

their substrates in aggregated than in monomolecular forms. The molecular mechan-

isms of this so-called interfacial activation has been the subject of controversial

debate, mainly performed on secretory PLA 2 (reviewed in Scott and Sigler,

1994; Gelb et al., 1995). In essence, there are two conceptions that are not mutually

exclusive and possibly even both effective. The ‘substrate model’ attributes the ac-

tivation by interfaces to the orientation, conformation or hydration states of the phos-

pholipid molecules in the aggregates, facilitating substrate diffusion from the inter-

facial binding surface to the active site (Scott et al., 1990b). According to this model,

activation must not be connected with conformational changes of the enzyme struc-

ture. It is supported by comparison of crystal structures of PLA 2 in the absence and

presence of substrate analogs, uncovering no significant structural modifications of

the enzyme (reviewed in Scott and Sigler, 1994). In contrast, the ‘enzyme model’ is

based on studies by NMR, fluorescence and also crystallographic measurements

indicating some conformational changes at lipid binding (reviewed in Scott and

Sigler, 1994; Kilby et al., 1995; Van den Berg et al., 1995a). Regions that are flexible

in the free enzyme, such as theN-terminal region and a surface loop become more

ordered in the presence of a substrate analog.

Several approaches have attempted to model the interface-activated kinetics. Ver-

ger et al. (1973) generally formulated the principle of enzyme activation by inter-

faces according to Equation (1), where the binding to the interface converts the

enzyme E to its activated form E*. This binds a substrate molecule and forms

the product P in a normal catalytic way. The model has been elaborated for a reac-

tion of PLA 2 at a monolayer.

E! E
!

þS

E
S!E
P! E
þP ð 1 Þ

The ‘surface dilution model’ (Deems et al, 1975; Dennis, 1994)includes one acti-

vating substrate molecule into the reaction equation [Equation (2)]. It is based on the

assumption that the water-soluble enzyme (E) first binds to one phospholipid mo-

lecule (S), constrained in an interface, to form ES. A second phospholipid molecule

is then bound in the catalytic site and forms the Michaelis complex ESS, from which

P is formed via the complex ESP.

EþS! ES!

þS

ESS!ESP! ESþP ð 2 Þ

12.4 Kinetic particularities of phospholipases and their consequences 235