Esterase , active site study

Gold and Linder (17) studied the esterase catalyzed hydrolysis of A-(-)-acetoxymethyl-(l-phenylethyl)nitrosamine. They found that the stereochemistry of 1-phenylethanol produced in the reaction was the same as that observed in the base catalyzed hydrolysis of the nitrosamine and also of N-(l-phenylethyl)nitrosocarbamate. These results indicated that the same diazotate was produced in all three reactions. The fact that no irreversible inhibition of the enzymatic hydrolysis of the nitrosamine was observed, while extensive irreversible inhibition was obtained with the nitrosocarba-mate, led these workers to conclude that the a-hydroxynitro-samine produced by the hydrolysis had sufficient stability to diffuse away from the active site of the enzyme. 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

The carboxypeptidases are released from their inactive precursors in the pancreatic juice of animals. The most studied example is bovine carboxypeptidase A, which contains one mole of zinc per protein molecular weight of 34 500. These enzymes cleave the C-terminal amino acid residue from peptides and proteins, when the side-chain of the C-terminal residue is aromatic or branched aliphatic of l configuration. At least the first five residues in the substrate affect the activity of the enzyme. The enzyme also shows esterase activity. Esters and peptides inhibit each other competitively, indicating that the peptidase and esterase sites overlap, even if they are not the same. 

The role of certain residues in the enzyme mechanism has been confirmed by chemical modification studies, notably for tyrosine. 14 Modification of tyrosyl residues (for example acetylation or nitration) leads to loss of peptidase activity and enhancement of esterase activity. The presence of the inhibitor -phenylpropionate protects two tyrosine residues from acetylation. Those are Tyr-248 and probably Tyr-198, which is also in the general area of the active site. The modified apoenzyme has lower affinity for dipeptides, as might be expected from the loss of hydrogen bonding between Tyr-248 and the peptide NH group. 

Substrates bind to P-gp while they are associated with the plasma membrane this process is possibly the most important aspect of P-gp-mediated efflux activity to appreciate. By using fluorescent dye esters, it was shown that P-gp interacts with its substrates within the plasma membrane. As these dye esters cross the membranes, esterases quickly hydrolyze the esters to their free acid form in the cytoplasm. Cells expressing P-gp showed no accumulation of the free acid dye in the cytoplasm clearly illustrating that P-gp can efflux substrates directly from the membrane (129). Additionally, P-gp can bind to substrates at the inner leaflet—cytosolic interface as demonstrated in studies with the P-gp substrate rhodamine 123 (133). It was shown that P-gp does not influence drug concentration in the exofacial leaflet (134), thus implying that P-gp only binds compounds from either within the inner leaflet or at the inner leaflet—cytosolic interface. These findings clearly show that the behavior of the substrate/inhibitor within the lipid barrier is likely to be a primary determinant of P-gp-mediated efflux activity. This separates P-gp from traditional transporters in which binding of the substrate to the active site in an enzyme-like fashion is the primary determinant of transport activity. 

According to this model, the action of the peptide catalysts used in the Julid-Colonna epoxidation bears a lot of similarity to that of enzymes, in particular the binding/activation and proper orientation of the substrates which ultimately effects the excellent enantioselectiv-ities in the overall process. In fact, the H-bonding motif discovered as the catalytically active site also acts as the oxy-anion hole in serine esterases and is known to bind/stabilize a variety of fully or partially negatively charged entities (Milner-White and Watson 2002a,b), the P-hydroperoxyenolate, in the present case. Studies by Roberts, Kelly,... 

Pig Liver Esterase (PLE). This is the more used car-boxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1, CAS 9016-18-6) which physiologically catalyzes the hydrolysis of carboxylic acid esters to the free acid anion and alcohol. PLE is a serine hydrolase which has been widely used for the preparation of chiral synthons and these applications have been fully reviewed. An active-site model for interpreting and predicting the specificity of the enzyme has been published. In the pioneering studies of the enzyme applications field, PLE was used for the chiral synthesis of mevalonolactone. Prochiral 3-substituted glutaric acid diesters... 

In the presence of human serum albumin, the H spectrum of acetyl-salicyclic acid is specifically shifted and broadened [119]. The interpretation of changes in T, and T2 require several theoretical assumptions. These have been discussed in detail [120] for JV-acetylsulphanilamide and acetate binding to the active site of carbonic anhydrase. It was concluded that the acetyl groups of these inhibitors have a motion additional to that of the enzyme. It can be shown by NMR that acetate binds to two sites on the enzyme, only one of which is inhibitory to esterase activity (methyls are 4.3 and 4.8 A from the metal in the Mn substituted enzyme [121]). Strict care must be taken to avoid paramagnetic impurities when NMR relaxation enhancement by diamagnetic macromolecules is being studied. A preparation of carbonic anhydrase, for example, can contain 0.24 paramagnetic Cu atoms per Zn atom [122]. 

The earliest observation that implied evolutionary links between all lipases was that of the consensus pentapeptide G-X-S-X-G, subsequently shown to contain the nucleophilic serine. The apparent similarity of this sequence to that found around the active serine in the chy-motrypsin and subtilisin families of serine proteinases prompted a number of authors to infer an evolutionary relationship between the three families. Further evidence in support of such a link came from secondary structure prediction studies indicating that the nucleophilic serine in a lipase is most likely within a /3 turn, structurally reminiscent of proteinases (Reddy et ai, 1986). In fact, one of the commonly used phrases found in introductions to many papers dealing directly or indirectly with lipases refers to the consensus G-X-S-X-G pentapeptide found in the active site of all serine proteinases and esterases. We now know that the implication that homology and/or structural similarities exist between the enzymes belonging to these diverse groups is incorrect. The matter has been dealt with in the literature (Derewenda and Derewenda, 1991 Liao et ai, 1992), but it seems appropriate to review some of the conclusions. 

While A-esterase(s) and B-esterases interact kinet-ically with paraoxon in a similar fashion (Figure 3), the molecular events occurring at their active sites during catalysis are probably very different. The active site of B-esterases such as acetylcholinesterase has been well characterized and contains a serine residue that is phosphorylated by paraoxon at the hydroxyl group. In contrast, the active site of A-est-erase(s) has not been studied as extensively, but it likely does not contain a serine residue that participates in the hydrolysis of paraoxon. Additionally, A-esterase(s) requires a divalent cation like calcium for activity, whereas B-esterases do not. 

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. 

The importance of active site volume is also evident in the decreasing stere-ospecificity of esterases as the volumes of their active sites increase. In site-specific mutagenesis studies of mammalian AChE, Taylor et al. observed that the stereoselectivity of AChE was reduced 3-fold and 230-fold by substitution of small aliphatic groups for phenylalanine at positions 295 and 297, respectively. Eurthermore, in a comparison of the stereoselectivity of AChE, BChE, and CaE, whose relative active site volumes are 3 5 30, the reported ratio of reaction rates of C(+)P(-) and C(+)P(+) stereoisomers of soman for AChE, BChE, and CaE are 17500, 290, and 135, respectively. " Even though the stereospecificity of CaE is reduced by its larger active site volume in comparison to ChE, it still maintains a 135-fold greater reactivity with the most toxic stereoisomers [i.e., C(+)P(-) soman]. 

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