Keywords
Lipases, Cysteine-lipases, Catalytic triad, Enzyme activity, Digestive tract, Immunological response
Commentary
Lipases, esterases and proteases constitute superfamilies of hydrolases not only play an important role in the immune system, but also as catalysts in biotechnology and organic chemistry. Mechanistically, they all involve a similar catalytic triad. The mechanism of lipasecatalysis is defined by the catalytic triad Ser-His-Asp in which activated serine adds nucleophilically to the carbonyl function of an ester or lactone substrate in the rate-determining step with formation of a short-lived oxyanion which then fragments into an alcohol and a covalent acyl-enzyme intermediate, the latter rapidly undergoing reaction with water and liberating the respective carboxylic acid (Figure 1) [1-5].
In contrast, both serine-proteases with Ser-His-Asp as the catalytic triad and cysteine-proteases characterized by Cys-His-Asp have been identified as enzymes with high activity. It has also been shown that in the case of serine proteases, mutation to the respective cysteine-proteases leads to a partial or complete breakdown of activity, and the same applies to the opposite scenario in which a cysteine-protease is mutated into a serine-protease, which has led to lively discussions concerning the origin of these effects [6-12]. Recently, we demonstrated that the conversion of a serine-lipase into an artificial cysteinelipase also induces significant loss of activity [13].
Today, it is well known that the human digestive tract has a prominent influence on the immune system. As already alluded to in the above short introductory information, lipases play an important role in a number of health problems. Only a few typical studies are cited here, the focus in these cases being on such enzymes as monoacylglycerol lipases, triglycerol lipases, and phospholipases [14-26]. In these and other works available in the extensive literature, sequence information was generally presented, showing that serine-lipases are indeed involved; in some studies, this was just assumed by the authors and no mention of possible cysteinelipases as alternatives was made.
In our study describing the transformation of a serineto a cysteine-lipase, Candida antarctica lipase B (CALB) was used as the model hydrolase [13]. The catalytic triad of this standard and in biotechnology often applied lipase is Asp187-His-224-Ser105 (Figure 1) [1-5,27-28]. In our study, mutant Asp187-His-224-Cys105 as a cysteine-lipase was shown to have a very low activity for a number of structurally different substrates. In order to regain and perhaps even to surpass the activity of wildtype (WT) CALB in a model transformation involving the hydrolytic kinetic resolution of a racemic ester, we utilized the techniques of directed evolution [29-32]. Specifically, saturation mutagenesis at rationally chosen residues surrounding the binding pocket according to the Combinatorial Active-site Saturation Test (CAST) [33] was performed, followed by Iterative Saturation Mutagenesis (ISM) [33-34] at other hotspot residues around the binding pocket [13]. The best evolved mutant, W104V/S105C/A281Y/A282Y/V149G, showed a 40-fold enhancement of activity in the model reaction, and was even more active than WT CALB in the hydrolysis of further substrates.
The combination of X-ray structures, kinetics, molecular dynamics (MD) simulations and QM/MM computations revealed dynamic effects upon going from cysteine-CALB to the best mutant [13]. It was shown that the three additional mutations cause, inter alia, the re-adjustment of the now active catalytic triad Asp187-His-224-Cys105 in a way that produces the zwitterion Cys105-/His224+, thereby enforcing a novel 2-step mechanism rather than the traditional concerted addition process [13]. Changing the mechanism of an enzyme by introducing mutations is a rare event in protein engineering. The overall results demonstrate that cysteine-lipases can in fact be active, but they do not prove that such lipases occur in nature.
The distinction between lipases and esterases continues to be a subject of considerable interest. Traditionally it was believed that lipases have a lid which opens upon interaction with a hydrophobic substrate by interfacial activation, in contrast to esterases [1-5,35-36]. However, uncertainties remain, and a sharp distinction may not matter [37-38]. In view of our recent study [13], we believe that an intensive search for natural cysteine-lipases (or esterases) could be a fruitful venture in different areas of lipase research, immunology and virology.
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