Deacylation step

Hydrolysis of esters and amides by enzymes that form acyl enzyme intermediates is similar in mechanism but different in rate-limiting steps. Whereas formation of the acyl enzyme intermediate is a rate-limiting step for amide hydrolysis, it is the deacylation step that determines the rate of ester hydrolysis. This difference allows elimination of the undesirable amidase activity that is responsible for secondary hydrolysis without affecting the rate of synthesis. Addition of an appropriate cosolvent such as acetonitrile, DMF, or dioxane can selectively eliminate undesirable amidase activity (128). 

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

Table 12. Enantioselectivities in the acylation and deacylation steps in the burst kinetics of the reaction of (Z)-Phe-PNP(52)...

The ratios of these slopes for L- and D-esters are shown in Table 12. The kL/kD values of the acylation step in the CTAB micelle are very close to those in Table 9, as they should be. It is interesting to note that the second deacylation step also occurs enantioselectively. Presumably it is due to the deacylation ocurring by the attack of a zinc ion-coordinated hydroxide ion which, in principle, should be enantioselective as in the hydroxyl group of the ligand. Alternatively, the enantioselectivity is also expected when the free hydroxide ion attack the coordinated carbonyl groups of the acyl-intermediate with the zinc ion. At any rate, the rates of both steps of acylation and deacylation for the L-esters are larger than those for the D-esters in the CTAB micelle. However, in the Triton X-100 micelle, the deacylation step for the D-esters become faster than for the L-esters. 

The first step, which is called the acylation reaction, involves a formation of an acyl-enzyme where the RC(0 )X group is covalently bound to the specially active serine residue and the XH group is expelled from the active site. The second step, which is called the deacylation step, involves an attack of an HY group on the acyl-enzyme. Here we concentrate on the acylation step which is the reverse of the second step when X and Y are identical. 

Fig. 21 Rate-pH dependence of the acylation and deacylation steps in the chymotrypsin-catalysed hydrolysis of 4-nitrophenyl trimethylacetate...

Serine peptidases can hydrolyze both esters and amides, but there are marked differences in the kinetics of hydrolysis of the two types of substrates as monitored in vitro. Thus, the hydrolysis of 4-nitrophenyl acetate by a-chy-motrypsin occurs in two distinct phases [7] [22-24]. When large amounts of enzyme are used, there is an initial rapid burst in the production of 4-nitro-phenol, followed by its formation at a much slower steady-state rate (Fig. 3.7). It was shown that the initial burst of 4-nitrophenol corresponds to the formation of the acyl-enzyme complex (acylation step). The slower steady-state production of 4-nitrophenol corresponds to the hydrolysis of the acetyl-enzyme complex, regenerating the free enzyme. This second step, called deacylation, is much slower than the first, so that it determines the overall rate of ester hydrolysis. The rate of the deacylation step in ester hydrolysis is pH-dependent and can be slowed to such an extent that, at low pH, the acyl-enzyme complex can be isolated. 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

Furthermore, the 3D-location of Tyr150 is quite different from that of Glu166 in class-A /3-lactamases. The observed resistance of the methoxylated cephalosporins to class-C /3-lactamases is due to a slow deacylation step [35], This inhibition is the result of the formation of an acyl-enzyme intermediate... 

In order to recover both amines in ophcaUy achve form the amide is hydrolyzed chemically by reachon with NaOH in aqueous ethylene glycol at 150 °C. This brute force method would certainly lead to problems with amines containing other functional groups and is in stark contrast to the elegant enzymatic procedure used for the first step. Hence, an overall greener process can be obtained by employing an enzymatic deacylation step in what we have called an easy-on/easy-off process... 

Both acylation and deacylation can be performed with a lipase as shown in Scheme 6.9 in which a CALB CLEA was used in the deacylation step that, not surprisingly, was rather slow compared to deacylation with pen acylase. 

Reaction Mechanism of Lipases and Implications for Monomer Acceptance in the Acylation and Deacylation Step... 

Fig. 1 Catalytic mechanism of CALB showing an acylation and deacylation step and the formation of a covalently bound acyl-enzyme intermediate bottom right) [16]...

The natural substrates of lipases are triglycerides and, in an aqueous environment, lipases catalyze their hydrolysis into fatty acids and glycerol. In anhydrous media, lipases can be active in the reverse reaction [19]. In fact, in the acylation step, acids, lactones, (cyclic) carbonates [20, 21], cyclic amides [22, 23], (cyclic) thioesters [24, 25], and cyclic phosphates [26] have been found to act as suitable acyl donors. In the deacylation step, apart from water, lipases also accept alcohols [27], amines [28, 29], and thiols [30] as nucleophiles although the specificity of lipases is lower for amines and thiols than for water and alcohols [31]. 

Although many publications have covered the enantioselectivity of lipases in the deacylation step, their enantioselectivity in the acylation step (i.e., towards the acyl donor) has received much less attention. Generally, the selectivity of lipases towards racemic esters or acids is low to moderate [75-77]. Directed evolution and site-directed mutagenesis lead to a significant increase in the selectivity of the wild-type enzymes [78-80]. However, the enantiomeric ratios attained are still well below those typically obtained in kinetic resolutions of secondary alcohols. 

The first enzymatic polymerizations of substituted lactones were performed by Kobayashi and coworkers using Pseudomonas fluorescens lipase or CALB as the biocatalyst [90-92]. A clear enantiopreference was observed for different lactone monomers, resulting in the formation of optically active polymers. More recently, a systematic study was performed by Al-Azemi et al. [93] and Peelers et al. [83] on the ROP of 4-alkyl-substituted CLs using Novozym 435. Peelers et al. studied the selectivity and the rates as a function of the substituent size with the aim of elucidating the mechanism and the rate-determining step in these polymerizations. Enantio-enriched polymers were obtained, but the selectivity decreased drastically with the increase in substituent size [83]. Remarkably for 4-propyl-e-caprolactone, the selectivity was for the (R)-enantiomer in a polymerization, whereas it was S)-selective in the hydrolysis reaction. Comparison of the selectivity in the hydrolysis reaction (Fig. 10b) with that of the polymerization reaction (Scheme 8a) revealed that the more bulky the alkyl substituent, the more important the deacylation step becomes as the rate-determining step. 

In the catalytically active complex 4-Ba the negative poles and the polyether bridge act as working units that perform cooperatively in providing the driving force for the formation of the complex itself, whereas the metal ion serves as an electrophilic catalyst both in the acylation and deacylation steps. The crucial importance of the polyether bridge is demonstrated by the disappearance of any catalytic activity upon replacement by two methoxy groups. 

Analysis ofthe kinetic data shows that the barium salt of 7, as well as the analogous salts of its higher homologues, perform much less efficiently than 4-Ba. The Ba complex of 7 turns over with a very low efficiency, caused by the extreme slowness of the deacylation step. Only a minor fraction ofthe liberated pNPOH in the steady-state phase is due to the expected double displacement mechanism. A larger fraction is most likely ascribable to the metal ion not sequestered by 7, and thereby available in solution for electrophilic assistance to direct methanolysis of the ester reactant. 

Step 3 Second nucleophilic reaction, release of remainder of substrate (dealkylation or deacylation step) ... 

The thematic approach to isolating the deacylation step is to generate the acylen-zyme in situ in the stopped-flow spectrophotometer by mixing a substrate that acylates very rapidly with an excess or stoichiometric amount of the enzyme. The acylenzyme is formed in a rapid step that consumes all the substrate. This is then followed by relatively slow hydrolysis under single-turnover conditions. For example, acetyl-L-phenylalanine p-nitrophenyl ester may be mixed with chy-motrypsin in a stopped-flow spectrophotometer in which the enzyme is acylated in the dead time. The subsequent deacylation may be monitored by the binding of proflavin to the free enzyme as it is produced in the reaction.8... 

The deacylation step. Listed in Table 16.2 are data for the deacylation of various acylenzymes. (Further values for amino acids were given in Table 7.3). The most reactive derivative is that of acetyl-L-phenylalanine. As was discussed... 

The strategy is to measure the rate constants k2 and k3 of the acylenzyme mechanism (equation 7.1) and to show that each of these is either greater than or equal to the value of kCM for the overall reaction in the steady state (i.e., apply rules 2 and 3 of section Al). This requires (1) choosing a substrate (e.g., an ester of phenylalanine, tyrosine, or tryptophan) that leads to accumulation of the acylenzyme, (2) choosing reaction conditions under which the acylation and deacylation steps may be studied separately, and (3) finding an assay that is convenient for use in pre-steady state kinetics. The experiments chosen here illustrate stopped-flow spectrophotometry and chromopboric procedures. 

---