Oxyanion hole esterases

Clearly, the oxyanion hole is now as significant a feature of the binding site of such acyl transfer abzymes as it is already for esterases and peptidases — and not without good reason. Knossow has analysed the structures of three esterase-like catalytic antibodies, each elicited in response to the same phosphonate TSA hapten (Charbonnier et al., 1997). Catalysis for all three is accounted for by transition state stabilization and in each case there is an... 

Later, oxyanion holes were also discovered in other proteases, such as the cysteine protease papain, and in esterases and lipases, enzymes capable of esterification or ester hydrolysis. Interestingly, in these esterases, sometimes up to three hydrogen bond donors can be located within 3 A of the carbonyl oxygen atom, whereas such triple hydrogen bonding motifs have not yet been found in the proteases. 

All of these esterases appear to act by mechanisms closely related to those of proteases. Acetylcholinesterase contains an active site serine that reacts with organophosphorus compounds (Box 12-E) and is part of an Asp-His-Ser catalytic triad which lies in a deep "gorge" as well as an oxyanion hole.637 A surprise is the absence of an essential carboxylate group that might bind the positively charged trimethylammonium... 

A peptide (73 residues) which has chymotrypsin-like esterase activity has been designed and synthesized by Hahn et al. (1990). The peptide consists of a bundle of four short helical sequences, and the catalytic residues were synthesized at the amino end of the bundle in spatial relationships similar to chymotrypsin. Also, the oxyanion hole and substrate binding pocket for acetyltyrosine ethyl ester, a chymotrypsin substrate, have been included in the design, the peptide cleaves this ester at a rate that is 1/100 of the chymotrypsin rate. It is also inhibited by chymotrypsin inhibitors. 

Figure 9.15 Carboxypeptidase IL The structure of carboxypeptidase II from wheat (right) is illustrated with its two chains (blue and red). Notice that the catalytic triad of carboxypeptidase II (left) is composed of the same amino acids as those in chymotrypsin, despite the fact that the enzymes display no structural similarity. The residues that form the oxyanion hole are highlighted in yellow. This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. All these enzymes make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. [Drawn from IWHS.pdb.]...

CE Family 1 is very large and contains members which do not act on carbohydrate-derived substrates. The crystal structure of a CE 1 domain of XynlOB modular enzyme from Clostridium thermocellum has been solved. " The CE 1 domain is a feruloyl esterase which hydrolyses the feruloyl groups attached to some arabinofuranosyl 05 groups in native xylan. (The Xyn lOB protein as a whole consists of two CBM 22 domains, a dockerin domain, and a GH 20 xylanase domain, and forms part of a cellulosome - see Section 5.10.) The enzyme has the common a/p hydrolase fold. Studies of ferulic acid complexes of the inactive alanine mutant of the active site serine revealed the classic catalytic triad, and two main-chain peptide NH bonds are in place to form an oxyanion hole . A remarkable feature is that the enzyme as repeatedly isolated was esterilied on the active site serine by phosphate or sulfate. 

CE 6 apparently contains only acetyl xylan esterases, but catalytic functions of many of the proteins in it have not been experimentally confirmed. One such protein from Arabidopsis thaliana proved to have a catalytic triad,and as isolated was found to have the catalytic serine covalently modified by PMSF. This enabled an oxyanion hole formed by two main-chain NH and a side-chain carboxamide to be identified. 

CE 12 contains serine enzymes which are likewise deacetylases. The rham-nogalacturonan I acetyl esterase of Aspergillus aculeatus removes acetyl groups from the 2- and 3-positions of the main-chain galacturonic acid residues of rhamnogalacturonan I it is a serine esterase, with a Ser-His-Asp catalytic triad and an oxyanion hole made from two main-chain peptide NHs and an Asn side-chain, but does not have the ot/p fold. 

Summarize the roles of the catalytic triad in the mechanism of chymotrypsin and the relationship of the oxyanion hole to the tetrahedral intermediate of the reaction. Appreciate that these features are present in other proteases, esterases, and lipases. 

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