Acyl 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). 

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... 

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. 

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. 

MECHANISM FIGURE 6-21 Hydrolytic cleavage of a peptide bond by chymotrypsin. The reaction has two phases. In the acylation phase (steps to ), formation of a covalent acyl-enzyme intermediate is coupled to cleavage of the peptide bond. In the deacylation phase (steps to ), deacylation regenerates the free enzyme this is essentially the reverse of the acylation phase, with water mirroring, in reverse, the role of the amine component of the substrate. Chymotrypsin Mechanism... 

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 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. 

In discussing possible mechanisms for the reactions catalyzed by E. coli glutaminase in Section I, it was concluded that either a two-step acylation-deacylation pathway or a one-step route, displacement by the ultimate nucleophile, could be accommodated by the results. It may be noted that any single displacement mechanism for a group transfer reaction requires that both incoming and outgoing substituent groups associate with the enzyme at the same time... 

It was found that 46 behaves as an exceptional substrate of trypsin, showing a the reaction mode which had not been observed before. Fig. 3 shows the time course of the tryptic catalysis of 46 monitored by the amidinophenol liberation under the condition that the substrate is in much higher concentration than the enzyme. After rapid mixing of enzyme and substrate, a rapid acylation step is observed and a slow deacylation then follows. The kinetics follow a Michaelis-Menten equation strong binding affinity, efficient acylation, and rate-determining slow deacylation steps, which are exactly the same as those of normal-type substrates. As a result, the accumulation of the acyl enzyme intermediate (EA) is realized in the course of the steady-state hydrolysis [cf. Eq. (6)]. 

The hydrolyses of the neutral substrates by partially alkylated poly(4-vinyl-pyridine) (I) were studied (63, 64). The rate enhancement is attributed to the hydrophobic interaction between the substrate and the polymer. In this hydrolysis, the rate of the substrate decomposition and the rate of the catalysis hold almost constant. It was explained that the deacylation step in the hydrolysis is affected by the bulkiness of the alkyl group in the polymer in which the alkyl group interferes an attack of water molecule on the acylated intermediate. 

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