Esterase substrate rules

Some of the general rules for substrate-construction are the same as those for esterase-substrates (Scheme 2.21), such as the preferred close location of the chirality center and the necessity of having a hydrogen atom on the carbon atom bearing the chiral or prochiral center. However, other features are different ... 

A rule, similar to Prelog s rule, has been proposed for the enzyme-mediated hydrolysis of the esters of secondary alcohols. Esters of the enantiomers 31 usually react faster. This rule correctly predicted the configuration of 14 out of 15 substrates when cholesterol esterase was used, 63 out of 64 substrates with a lipase from Pseudomonas cepacia, and of 51 out of 55 cyclic substrates using a lipase from Candida rugosa24°. 

As mentioned earlier (Section 4.2.1.1), empirical rules for the enantioselectivity of hydrolases have been developed. It is important to keep in mind that these rules do not work for all substrates. Most rules are based on pockets, which indicate how the steric bulk of the substituents in the substrate fit into the environment of the active site. Thus, such rules have been suggested for pig liver esterase(PLE) [66], the protease subtilisin [66-68], and certain lipases [69-71]. For secondary alcohols, most lipases follow the simple rule of Kazlauskas, which was developed for Pseudomonas cepacia, and which is depicted in Figure 4.4 [72]. This model implies that the fast-reacting enantiomers binds to the active site as described in Figure 4.4, whereas the slowly reacting one is not able to achieve a comfortable fit, because it will require the large substituent L to fit into the smaller pocket. In contrast to lipases, subtilisin displays opposite enantioselectivity toward secondary alcohols [68]. 

The stereospecificity may be carried, either by the carboxylic acid moiety, or by the alcohol part of the molecule. There is no rule up to now to predict if a given molecule will be a substrate, and if the enzyme will express its stereospecificity toward it. Screening of lipases and esterases is the only method ta select firstly the active enzymes, and secondly the specific ones that give the wanted isomer. 

The replacement of water by an organic solvent is almost invariably accompanied by a dramatic decrease in the catalytic activity of an enzyme and a decline in its substrate specificity. Thus, significant activities in non-aqueous media are the exception rather than the rule (35, 36). Surprisingly, the esterase-lipase preparation showed very good esterification properties and was tested extensively in a model system consisting of oleic acid and ethanol. Equimolar quantities of oleic acid and ethanol undergo rapid esterification when stirred at room temperature... 

The structural features of more than 90% of the substrates which have been transformed by esterases and proteases can be reduced to the general formulas given in Scheme 2.21. The following general rules can be applied to the construction of substrates for esterases and proteases ... 

CALB is an exceptionally robust protein which is deactivated only at 50-60°C, and thus also shows increased resistance towards organic solvents. In contrast to many other lipases, the enzyme appears to be rather rigid and does not show a pronounced effect of interfacial activation [430], which makes it an intermediate between an esterase and a lipase. This latter property is probably the reason why its selectivity could be predicted through computer modeling to a fair extent [431], and for the majority of substrates the Kazlauskas rule (Scheme 2.49) can be applied. In line with these properties of CALB, selectivity-enhancement by addition of water-miscible organic cosolvents such as t-butanol or acetone is possible - a technique which is rather common for esterases. All of these properties make CALB the most widely used lipase both in the hydrolysis [432-437] and synthesis of esters (Sect. 3.1.1). 

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