Lipases conformational changes

Lowering the temperature in the lipase-catalyzed resolution usually enhances the enantioselectivity. The phenomenon does not come from the temperature-induced conformational change of lipase, but it is understandable on the basis of the theory of physical organic chemistry as explained below. ... 

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]. 

When water molecules interact with an enzyme, it is natural that conformational changes can occur, which in turn can cause changes in the selectivity of the enzyme. Since enantioselectivity of enzymes is of major importance for many applications, it is a common task to investigate how to choose reaction conditions providing the maximal enantioselectivity. As might be expected, because water can interact with enzymes in many ways, it is difficult to generalize the effects. In some studies of lipase-catalyzed esterification reactions, no effects of water activity on enantioselectivity were observed [30]. In a similar study, no effects were observed in most cases, while the enantioselectivity of one lipase-catalyzed reaction decreased... 

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. 

Figure 32 includes results illustrating the performance of lipase/car-bon monolith systems in an acylation reaction. For comparison, the free lipase and a commercial immobilized lipase (Novozyme) were also tested. As expected, in all cases the specific activity of immobilized lipase was foimd to be lower than that of the free enzyme. Such a difference is usually ascribed to conformational changes of the enzyme, steric effects, or denaturation. For the monolithic biocatalysts, the activity of the immobilized catalyst relative to that of the pure enzyme was found to be 30-35%, and for the Novozyme catalyst about 80% in the first rim. However, the Novozyme catalyst underwent significant deactivation, in contrast to the carbon monolith-supported catalysts. The deactivation of the Novozyme catalyst in consecutive runs is probably a consequence of the instability of the support matrix under reaction conditions (101,102). 

Solvent Type The affect of different organic solvents on various enzyme catalytic activities has been demonstrated (37). From a mechanistic standpoint, the effect of organic solvents on enzyme catalysis is still debated (38). Conformational changes in enzymes, when suspended in organic solvents, have been reported to result in alteration of substrate specificity and affinity of substrates for enzymes (39). The polarity of organic solvents affects lipase-catalyzed reactions as do other variables that are critical to enzyme activity in organic media. The nature of the... 

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 general terms, the crystallographic results show that lipases contain several distinct sites, each responsible for a specific function. The hydrolysis of the ester bond is accomplished by the catalytic triad, responsible for nucleophilic attack on the carbonyl carbon of the scissile ester bond, assisted by the oxyanion hole, which stabilizes the tetrahedral intermediates. The fatty acid recognition pocket defines the specificity of the leaving acid. There is also one or more interface activation sites, responsible for the conformational change in the enzyme. In this section the discussion is on the available structural data relevant to the function of all these sites. 

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. 

The water-mediated interactions may also be of considerable importance. In the activated form the lid occupies a new position on the surface of the molecule, some 8 A away from the original location. This deep surface depression extends 10 A into the molecule and is filled in the native enzyme by 18 water molecules, half of which are direcdy hydrogen bonded to the polar protein groups. During the conformational change ail but three of these solvent molecules are expelled. In the lipase-inhibitor complex these three molecules become buried and mediate the polar contacts formed primarily by Asn-87. 

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. 

Lipases are another important group of hydrolases. The most commonly used example is porcine pancreatic lipase (PPL). Lipases tend to function best at or above the solubility limit of the hydrophobic substrate. In the presence of water, the substrate forms an insoluble phase (micelles) the concentration at which this occurs is called the critical micellar concentration. The enzyme is activated by a conformational change that occurs in the presence of the micelles and results in the opening of the active site. Lipases work best in solvents that can accommodate this activation process. PPL is often used as a relatively crude preparation called pancreatin or steapsin. The active site in PPL has not been as precisely described as the one for PLE. There are currently two different models, but they sometimes make contradictory predictions. It has been suggested that the dominant factors in binding are the hydrophobic and polar pockets (sites B and C in Figure 2.29), but that the relative location of the catalytic site is somewhat flexible and can accommodate to the location of the hydrolyzable substituent. ... 

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