Mass mz

Fig. 9.15 MALDI-TOF mass spectra of HPL (I) or HPL incubated 1 h with orlistat (II) at a HPL:orlistat molar ratio of 1 : 100. Adapted from [112].

of orlistat (molecular mass, 496 Da) to HPL, as previously reported by Luthi-Peng et al. [105].

9.4.2.3 Kinetic Model Illustrating the Covalent Inhibition of HPL in the Aqueous Phase

On the basis of the above mentioned kinetic and mass spectrometry analysis, a model was proposed for the covalent inhibition of HPL by orlistat in the aqueous phase as well as its partial reactivation at the lipid-water interface (Fig. 9.16). This model takes into account the putative existence of two different forms of the covalent orlistat/lipase complex.

Fig. 9.16 Kinetic model illustrating the inhibition of HPL by orlistat in the aqueous phase and its reactivation at a lipid-water interface. The following symbols and abbreviations are used here: E, free enzyme (molecule/volume); E*, interfacial enzyme (molecule/surface); FA, fatty acid at the interface (molecule/surface); E*-FA, interfacial enzyme-fatty acid complex (molecule/surface); THLc, closed reactive orlistat in the bulk (molecule/volume); THLo, open non-reactive orlistat at the interface (molecule/surface); E-THLo^ form 1 of cova

Fig. 9.16 Kinetic model illustrating the inhibition of HPL by orlistat in the aqueous phase and its reactivation at a lipid-water interface. The following symbols and abbreviations are used here: E, free enzyme (molecule/volume); E*, interfacial enzyme (molecule/surface); FA, fatty acid at the interface (molecule/surface); E*-FA, interfacial enzyme-fatty acid complex (molecule/surface); THLc, closed reactive orlistat in the bulk (molecule/volume); THLo, open non-reactive orlistat at the interface (molecule/surface); E-THLo^ form 1 of cova lent enzyme-orlistat complex in the bulk (molecule/volume); £-THLo2, form 2 of covalent enzyme-orlistat complex in the bulk (molecule/volume); E*-THLo1, interfacial form 1 of covalent enzyme-orlistat complex (molecule/surface); E*-THLo2, interfacial form 2 of covalent enzyme-orlistat complex (molecule/surface). TAG, triacylglycerol at the interface (molecule/surface); DAG, diacylgly-cerol at the interface (molecule/surface). From [112].

One can hypothesize that the covalent binding of orlistat to HPL occurs in two possible ways. One fraction of the lipase molecules may form a covalent complex in the aqueous phase with the open /-lactone ring of orlistat in a given form (E-THLox), whereas the other fraction of the lipase molecules may also form a covalent complex with orlistat, but in another form (E-THLo2). The hypothesis that a fraction of the inhibited lipase molecules could form a non-covalent complex with orlistat in its closed form (E-THLc) can be ruled out, since the mass spectra analysis (Fig. 9.15) showed the presence of only the HPL-orlistat covalent complex.

In the lipase assay system, when the (E, THLc, E-THLo1 and E-THLo2) mixture was added to a TAG emulsion, the 'weaker complex (E-THLo1) was hydrolyzed, explaining the reactivation process as revealed by the existence of lag times. The hydrolysis of this 'weak' complex was enhanced when bile salts were present in the assay medium, probably due to the formation of a mixed orlistat/lipase/bile salt complex. The fact that the reactivation of HPL did not reach 100% of its initial value (before incubation with THL) at the end of the kinetic experiments (Fig. 9.13) might be attributable to the existence of a second fraction of lipase molecules, which is covalently and irreversibly inhibited by orlistat (E-THLo2). The coexistence of two different forms of orlistat covalently bound to HPL may be due to the inhibitor molecule being differently oriented in the catalytic cavity of the lipase molecule. The existence in the catalytic cleft of lipases of two different orientations in the case of molecules of substrate analogues was previously reported [113, 114].

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