Food Lipids: Chemistry, Nutrition, and Biotechnology

the active site and a larger hydrophobic site, and allow access of the substrate to the
active site. The working hypothesis for the mechanism of interfacial activation is
based on the three-dimensional structural analyses of lipases from Rhizomucor miehei
(88,103), human pancreatic lipase (81), G. candidum (90,91), and C. rugosa (92,104).
The two latter lipases, for example, are globular, single-domain proteins built around
an 11-stranded mixed � sheet with domains of 45 A˚ � 60 A˚ � 65 A˚ (105).
Activation of R. miehei lipase involves the movement of a 15 amino acid long
lid in a hinge-type, rigid-body motion that transports some of the atoms of a short
� helix by more than 12 A˚ (103). This, combined with another hinge movement,
results in the exposure of a hydrophobic area representing 8% of the total molecular
surface. In C. rugosa, comparison of open (92) and closed (104) conformation indicates
that activation of the lipase requires the movement and refolding, including
a cis-to-trans isomerization of a proline residue, of a single surface loop to expose
a large hydrophobic surface where the substrate likely interacts. Lid reorganization
contributes to the formation of a catalytically competent oxyanion hole and creation
of a fully functional active site (105). The scissile fatty acyl chain is bound in a
narrow, hydrophobic tunnel, where modeling studies suggest that the substrate must
adopt a tuning fork conformation (105,106). There is a tryptophan residue at the tip
of the lid (Trp 89 ) in the lipase from H. lanuginosa that plays an important role in
hydrolytic activity (107). When Trp 89 is substituted with other amino acids, activity
drops substantially and variably depending on the substituent amino acid.
It is difficult to visualize how the hydrophobic substrate molecule, which is
buried in the oil surface, gains access to the active site, which is buried within the
water-soluble lipase molecule. Blow (108) has suggested that upon activation, a
‘‘hydrophobic seal’’ forms at the interface that allows the substrate to enter the active
site without interacting with the bulk water; that is, the enzyme partially withdraws
the substrate molecule from the bulk oil with at least some of the acyl chains projecting
into the lipid.
Cygler et al. (105) have postulated that there are two tetrahedral intermediates
in the lipase-catalyzed hydrolysis of esters. Formation of a noncovalent Michaelis
complex between the lipase and triacylglycerol is followed by formation of a tetrahedral,
hemiacetal intermediate resulting from a nucleophilic attack by the serine O � .
The oxyanion resulting from formation of the enzyme–substrate complex is stabilized
by the amide groups of the oxyanion hole (e.g., Gly 123 in C. rugosa) and �
helix following the active site serine (105). Intermediate formation is followed by
cleavage of the substrate ester bond, breakdown of the tetrahedral intermediate to
the acyl enzyme, and protonation and dissociation of the diacylglycerol. The serine
ester of the acylated enzyme is attacked by an activated water molecule to form a
second tetrahedral intermediate, which is cleaved to give rise to the protonated enzyme
(serine residue) and fatty acid.
C. Activation/Inhibition
Calcium may stimulate lipase-catalyzed hydrolytic activity by (a) binding to the
enzyme resulting in a change in conformation, (b) facilitating adsorption of the lipase
to the substrate–water interface, and/or (c) removing from the interface fatty acid
products of hydrolysis that may reduce end-product inhibition of the reaction. Activation
of human pancreatic lipase by calcium is complex and variable depending
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