Interfacial conformational change

In 1958 Sarda and Desnuelle [79] discovered the lipase activation at the interfaces. They observed that porcine pancreatic lipase in aqueous solution was activated some 10-fold at hydrophobic interfaces which were created by poorly water-soluble substrates. An artificial interface created in the presence of organic solvent can also increase the activity of the lipase. This interfacial activation was hypothesized to be due to a dehydration of the ester substrate at the interface [80], or enzyme conformational change resulting from the adsorption of the lipase onto a hydrophobic interface [42,81,82]. 

More advanced semiempirical molecular orbital methods have also been used in this respect in modeling, e.g., the structure of a diphosphonium extractant in the gas phase, and then the percentage extraction of zinc ion-pair complexes was correlated with the calculated energy of association of the ion pairs [29]. Semiempirical SCF calculations, used to study structure, conformational changes and hydration of hydroxyoximes as extractants of copper, appeared helpful in interpreting their interfacial activity and the rate of extraction [30]. Similar (PM3, ZINDO) methods were also used to model the structure of some commercial extractants (pyridine dicarboxylates, pyridyloctanoates, jS-diketones, hydroxyoximes), as well as the effects of their hydration and association with modifiers (alcohols, )S-diketones) on their thermodynamic and interfacial activity [31 33]. In addition, the structure of copper complexes with these extractants was calculated [32]. 

A simple linear plot of the data allows AA to be obtained. The results of a set of experiments (Table 5) are surprising. AA is independent of the size or molecular weight of the protein. Although the cross-sections of the proteins studied range from 1000 to 10,000 A2, AA is nearly constant at 100 to 200 A2. Conclusion . . only a small portion of the protein molecule needs to enter the interface in order for adsorption to then proceed spontaneously (Ref.3), p. 290). It is as if only a small foothold or handhold is required to stabilize the molecule against desorption. Now firmly planted at the interface, the molecule can optimize its interfacial interactions by time-dependent orientation and perhaps conformational changes. The size of the foot is obviously relevant to the exchange discussion in Sect. 4.5. 

The interfacial layer is the inhomogeneous space region intermediate between two bulk phases in contact, and where properties are notably different from, but related to, the properties of the bulk phases (see Figure 6.1). Some of these properties are composition, molecular density, orientation or conformation, charge density, pressure tensor, and electron density [2], The interfacial properties change in the direction normal to the surface (see Figure 6.1). Complex profiles of interfacial properties take place in the case of multicomponent systems with coexisting bulk phases where attractive/repulsive molecular interactions involve adsorption or depletion of one or several components. 

True lipases show the interfacial activation phenomenon in their catalytic activity pattern. At low concentration of water-insoluble substrates, lipases are almost inactive, and the hydrolytic activity does not increase linearly. At a certain substrate concentration, however, the hydrolytic activity of lipases increases rapidly and the lipase kinetics resembles normal enzyme kinetics. This boost in activity is related to the formation of water-insoluble substrate aggregates such as micelles or another second phase. Only when this second phase is present, do lipases become fully active. This interfacial activation is caused by a large conformational change in the 3D structure of the lipases. In their water-soluble form, the active site is covered by a lid, which prevents the substrates from reaching it. At the lipidAvater interface, the lid is opened and the active site is accessible to the substrates. In addition, the now accessible area is mainly hydrophobic, which gives the open-form lipase the shape and behavior of conventional surfactant molecules with a hydrophilic and a hydrophobic moiety in one single molecule. 

Conflicting hypotheses were also put forward with respect to the mechanism of interfacial activation. Desnuelle et al. (1960) were the first to suggest that a conformational change in the enzyme could be responsible for the enhancement of activity at the oil-water interface. There were also other hypotheses. For example. Wells (1974) suggested that the apparent activation of lipases is due to the orientation of the scissile ester bond on the surface of micelles Brockerhoff (1968), on the other hand, pointed to the possibility of differences in solvation of the ester bond in solution versus a lipid phase, whereas Brockman et al. (1973) postulated that a steep substrate concentration gradient at the interface may provide an explanation. 

In lipases the existing database regarding the oxyanion holes is still limited. In RmL two amide groups (residues 145 and 146) were originally proposed as likely candidates for this function (Brady et al., 1990). However, structural analyses of the two RmL-inhibitor complexes (Brzozowski et al., 1991 U. Derewenda et al., 1992) revealed that the oxyanion hole is likely to be fully formed only after the conformational change associated with interfacial activation, and that it is made up of both the amide and the side-chain hydroxyl of Ser-82 (Fig. 6). A hy-... 

Since the pioneering studies of the phenomenon by Sarda and Des-nuelle (1958), many authors have hypothesized about the molecular basis of interfacial activation (see Section I). The present structural evidence supports the original proposal put forward by Desnuelle et al. (1960), who postulated a conformational change in the enzyme, fixing itself at the interface. All crystal structures of lipases clearly show that a change is necessary to expose the catalytic centers, which in the native enzymes are buried under various surface loops, or lids. 

A detailed analysis of the stereochemistry of the conformational changes associated with interfacial activation is possible for both RmL (Brzozowski et ai, 1991 U. Derewenda et ai, 1992) and hPL (van Til-beurgh et ai, 1993), although the low resolution of the latter study imposes some limitation on the accuracy of the observations. 

Mollmann, S.H., Jorgensen, L., Bukrinsky, J.T., Elofsson, U., Norde, W., and Frokjaer, S. (2006) Interfacial adsorption of insulin-conformational changes and reversibility of adsorption. European Journal of Pharmaceutical Sciences, 27,194—204. 

Lipases are interphase-active enzymes with hydrophobic domains. The hydro-phobic surface (loop) on lipase is thought to enable lipophilic interfacial binding with substrate molecules that actually induces the conformational changes in lipases. The open conformation will provide substrate with access to the active site, and vice versa. In certain types of lipases, the movements of a short a-hehcal hydrophobic loop in the lipase structure cause a conformational change that exposes the active sites to the substrate. This movement also increases the nonpolarity of the surface surrounding the catalytic site [30, 32, 34, 35]. Obviously, the hydrophobic surface plays an important role in the activity of lipase as an enzyme. 

Mollmann SH, Jorgensen L, BukrinskyJT, Elofsson U, Norde W, and Frokjaer S. Interfacial Adsorption of Insulin Conformational Changes and Reversibility of Adsorption. Eur JPharm Sci 2006 27(2-3) 194-204. 

It was noted that activity and conformation change with the amount of water inside micelles, pointing out the importance of the aqueous environment, easily adjustable as a function of the water content, which is impossible when studies are performed in aqueous solutions. Furthermore, because many properties (9) of the water core resemble those of water present at interfaces in biological systems, reversed micelles provide an excellent system for studying the interactions between polypeptides and interfacial water (10-11) or more generally their conformation when solubilized in micelles (12-15), depending on where the biopolymer is located inside the micelle and what its conformation is. 

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