PRINCIPALES CAUSAS DE OBESIDAD EN EL ECUADOR Y CÓMO

Lipases are enzymes which are characterised by their need for an insoluble substrate and hence require an interface. Lipases can act on soluble monomeric substrates but practical utilisation of lipase-catalysed reactions are restricted to situations where the overall substrate concentration is higher than its solubility in the natural solvent, water. This characteristic provides a convenient criterion for differentiating lipases from conventional esterases which usually act on only soluble monomeric substrates.

This feature was first observed by Sch(|)nheyder & Volqvartz, (1945) and later by Sarda & Desnuelle, (1958). It was demonstrated that in contrast to esterase, which exhibits a normal Michaelis-Menten activity dependence on substrate concentration, pancreatic

C hapters Lipases

lipase displayed no activity when a triacaproin substrate was in the monomeric state. However, when the solubility limit of the substrate was exceeded, there was an increased enzyme activity with the substrate in the emulsified state. The esterase appears to only be active on molecularly dispersed molecules whilst the lipase was capable of hydrolysing the substrate in an insoluble form. The observed rates of lipase-catalysed reactions were strongly influenced by the interfacial area available within the system (Desnuelle, 1961).

Many mechanisms have been proposed to explain this characteristic at either substrate and/or enzyme level but the actual pathway has yet to be elucidated. Theoretical interpretations o f the activation of lipases by interfaces have also been attempted by a number of authors. These can be divided into two groups: (i) those which assume that the substrates are activated by the presence of an oil/water interface, and (ii) those which assume that the lipase undergoes a change to an activated form upon contact with the oil/water interface (Verger & Haas, 1976).

It has been proposed that interfacial activation may be due to an increased concentration o f substrate molecules in the vicinity o f the interface which would cause a more ordered structuring of the lipid molecules resulting in more frequent enzyme-substrate collisions (Brockman et al, 1973). This would appear unlikely since esterases, which have a similar mode of action to lipases, behave differently. It has also been suggested that substrate aggregation and orientation into more suitable conformations for chemical reactions may influence enzyme catalysis. Substrate aggregation will cause a loss in rotational and translational energy resulting in a lower activation energy for lipolysis (Shah & Schulman, 1967; Mattson & Volpenhein, 1969; Brockerhoff, 1970 and Wells, 1974). Formation of substrate aggregates may also have a hydration effect on the lipid molecules which has been suggested may be required for lipase activation (Brockerhoff 1968).

The second theory involves the existence of separate adsorption and catalytic sites for the lipase such that the lipase can only become catalytically active after binding to the interface. The location and binding site has become known as the "Interface Recognition Site" and the active site as the "Classical Active Site". Associated with this is a conformational change to the lipase as it approaches the oil/water interface which aids

C hapters Lipases

the activating effect of substrate aggregation due to a higher degree of order in this region (Desnuelle et a l, 1960; James & Augenstein, 1966 and Dawson, 1969). It has been suggested that the orientation o f the enzyme is crucial since the lipase must orientate itself such that its active site is correctly located.

Orientation of the lipase has been considered of key importance with respect to both of the aforementioned theories. Studies by Mattson et aL, (1970) suggested that orientation of the lipase molecule at the oil/water interface and the specificity o f the enzyme for its substrate were the main factors determining the rate of hydrolysis. They also demonstrated that the oil/water interface may adsorb other proteins increasing competition for lipase adsorption thus influencing the rate of hydrolysis.

Brockerhoff (1969) also investigated orientation and the specific adsorption o f the lipase at the interface. It was proposed that the enzyme unfolds on adsorption which confers specificity by inducing conformational changes. It was also suggested that the Km, the Michaelis constant, may be the dissociation constant of the enzyme-interface complex and therefore, oil/water interfaces would behave as unspecific surfaces and adsorb proteins of the same affinity.

These differing results are probably justified since both the previous authors were using saturating interface concentrations where all the enzyme molecules are adsorbed onto the interface and hence, studied the affinity of the enzyme for its specific substrate in the interface. Therefore, the suggestion that orientation of the substrate molecules at the interface is important is probably valid.

At the oil/water interface, the lipid molecules will orientate in such a manner as to give rise to an ordered packing in which the polar heads are exposed to the water phase and the hydrocarbon tails are exposed to the organic phase. However, when lipid molecules are in true solution they orientate themselves randomly because they are completely surrounded by water molecules. This ordered continuum can also be achieved on the hydrophobic surface of a solid carrier. Rucka & Turkiewicz, (1990) concluded that only the immediate vicinity of the lipase should possess a hydrophobic character since the overall conformation of the enzyme and the local conformation of the active site depend

C hapters Lipases

primarily on the intramolecular forces and the short range interaction of the protein with the solvent.

Similarly, Malcata et a l, (1990) proposed that the true oil/water interface is not likely to be in direct contact with the immobilised lipase for this reason and the existence of lipolytic activity requires a continuous ordered hydrophobic microenvironment. The surfaces of contact of two phases provide a continuum irrespective of whether these two phases are liquid phases e.g. oil and water or a liquid and a solid phase e.g. oil and an immobilised lipase. This fact seems to contradict the generally accepted theory that an oil/water interface is required for lipases to exhibit catalytic activity.