Acyl-enzyme intermediate, possible

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

One possible mechanism for the hydrolysis of peptides or esters by carboxypeptidase A involves two steps with an anhydride (acyl-enzyme) intermediate.418 In the first step, the zinc(II) activates the substrate carbonyl group towards nucleophilic attack by a glutamate residue, resulting in the production of a mixed anhydride (127). Breakdown of the anhydride intermediate is rate determining with some substrates.419 An understanding of the chemistry of metal ion effects in anhydride hydrolysis is therefore of fundamental importance in regard to the mechanism of action of the enzyme. Until recently there have been few studies of metal ion-catalysed anhydride solvolysis. 

Clavulanic acid is a mechanism-based irreversible inhibitor and could be classed as a suicide substrate (Chapter 4). The drug fits the active site of (3-lactamase and the 13-lactam ring is opened by a serine residue in the same manner as penicillin. However, the acyl-enzyme intermediate then reacts further with another enzymic nucleophilic group (possibly NH2) to bind the drug irreversibly to the enzyme (Fig. 10.54). The mechanism requires the loss or gain of protons at various stages and an amino acid such as histidine present in the active site would be capable of acting as a proton donor/acceptor (compare the mechanism of acetylcholinesterase in Chapter 11). 

Applications of low temperature work in structural studies have been described in section 3(b). Application to enzyme action is best exemplified by the pioneering work of Fink and Ahmed [221] and Alber etal. [222] on elastase. JV-Carbobenzoxy-L-alanyl-p-nitrophenol ester was selected for study at — 55°C in a 70% methanol-water mixture. Kinetic studies in the presence of cryoprotectant enabled conditions for formation and stabilisation of the acyl-enzyme intermediate to be established. By monitoring changes in intensity of certain reflections as substrate flowed past the crystal at — 55°C, it was possible to show that the rate of formation of the acyl-enzyme was comparable to that obtained by monitoring p-nitrophenol release spectroscopically. The difference electron density map at 3.5 A resolution showed a peak consistent with the formation of an acyl-enzyme intermediate, but a detailed mechanistic interpretation requires higher resolution data. When the crystal was warmed to — 10°C and the data recollected, the peak in the difference synthesis disappeared, indicating that deacylation had occurred, consistent with the predictions from kinetic studies. 

For reactions in which a chromophoric product is released part way through the catalytic reaction—for example, in protease catalysis where an acyl-enzyme intermediate is formed—it is often possible to see the release of an equivalent amount of the product formed concurrently to the formation of the enzyme-substrate intermediate. For example, as shown in Figure 4, in the reaction of papain with N -carbobenzoxy-L-lysine p-nitrophenyl ester in 60% dimethyl sulfoxide at pH 6.1 (the pH optimum) a stoichiometric "burst of p-nitrophenol is observed at temperatures below — 40°C as the acyl-enzyme is formed, followed by no further release of p-nitrophenol, indicating that no turnover is occurring... 

Brief incubation of the enzyme at 0°C with radioactive acetyl-CoA led to the formation of acyl enzyme which could be isolated by chromatography on Sephadex [24]. The enzyme-substrate complex was then reacted with acetoacetyl-CoA with the concomitant formation of HMG-CoA. Further studies indicated that the functional group on the enzyme that accepted the acetyl residue was a cysteine sulfhydryl. 4 -Phosphopantetheine is known to accept acyl residues, but it was not found in this protein. The stoichiometry for acetylation was 0.7 acetyl groups per mole of enzyme since it is a dimeric protein with apparently identical subunits, this observation is surprising. Thus, it is possible that the subunits perform different functions for example, one could be regulatory. It is interesting to note that both the thiolase and HMG-CoA synthetase utilize acyl enzyme intermediates in their catalytic mechanisms. 

A potential substrate entryway (which presents anphipathic residues possibly to accommodate polar substrate head groups towards the FAAH active site) has been identified next to a-18 and a-19 helices, and it may indicate direct connection between the FAAH active site and the hydrophobic membrane bilayer. The mode for membrane binding of FAAH may facilitate movement of the FAA substrates directly from the bilayer to the active site, with no need for transport of these lipids through the aqueous cytosol. In this model, the substrate would first enter via the membrane to the active site following hydrolysis, the released fatty acid (hydrophobic) and amine (hydrophilic) products would then exit through the membrane-access and cytosolic-access channels, respectively. Moreover, the cytoplasmic port may serve the additional function of providing a way for a water molecule required for deacylation of the FAA-FAAH acyl-enzyme intermediate, which has been already characterized by LC-MS (Patricelli and Cravatt., 1999). 

Not only transpeptidation, but also a certain amount of peptide-peptide condensation, is possibly involved in the plastein reaction. With some of the proteases used for plastein formation, especially a-chymotrypsin (26, 52, 53, 54), the acyl-enzyme intermediate can be formed at pHs 5 from the reversal of the degradative reaction (Equation 1 E-OH + HOOC-CHR-NH E-O-OCCHR-NH- + H20). Once the acyl-enzyme intermediate is formed, the acyl group can be transferred to a nucleophile resulting in peptide bond synthesis. 

For more complex metabolites (phenols and tetraacetic acid lactone), further condensation of the triacetyl residue must take place and can only proceed if the priming acetyl residue, with its C-5 oxygen function, is stabilized away from the thioester region of the acyl-enzyme intermediate at this time. Possibly, the presence of NADPH assists in this process, in appropriate enzymes, since the ability to form triacetic acid lactone has been demonstrated by fatty acid and 6-methylsalicylate synthetases when deprived of this nucleotide, but other synthetases have not been tested for this effect. Moreover, the analogous formation of the styrylpyrone bisnoryangonin by flavan-one synthase (Kreuzaler and Hahlbrock, 1975a,b) confirms that the potential for synthesis of stable products with shorter chains does exist. 

In another study, Padovani showed that CALB-catalyzed modification of pendant ester groups of a polystyrene derivative provided a clear-cut regioselective transesterification reaction [152]. Of the pendant two ester groups available on the polymer backbone, only the ester group distant from the polymer backbone was involved in the lipase-catalyzed esterification reaction. The results suggested that formation of the acyl-enzyme intermediate is sterically not possible for the ester... 

The fit of the data to the plotted regression equation is obviously better in Fig. 5 than in Fig. 6. Of course, there are various biological factors that would affect how good a fit is achievable. These include (1) penetration of the antibiotic through the outer bacterial membrane to the sites of action, (2) reversible binding affinity of the antibiotic with the target enzymes, (3) rate of covalent attachment of the inhibitor molecules to the enzymes, and (4) rate of deacylation of the acyl-enzyme intermediate [see Eq. (1)]. These quantities have not been measured for the set of compounds treated here. Then, too, it is possible that the CNDO/2 MO... 

Some speculative, but necessarily incomplete comparison may be made for serine proteases. It is possible to distinguish a binding domain in proteases, the one located toward the C-terminus (C domain), but it is not possible to refer to the other domain as the catalytic one since it contains only two of the three catalytic residues of the charge relay system. Ser 195, which keeps a part of the substrate covalently bound in the acyl-enzyme intermediate pertains to the binding domain. 

If k2 > kj, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With -glucoside Aj from Asp. wentii and 4-nitrophenyl [ C]-2-deoxy-) -D-irra />jo-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-D-arabino-hexose bound to the same aspartate residue that had previously been labeled with the active-site-directed inhibitor conduritol B epoxide and with D-glucal." This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. 

Even lower temperatures have been used to study possible intermediate stages in the formation of the acyl enzyme. A tetrahedral intermediate (with a covalent bond between the substrate carbonyl carbon atom and the oxygen atom of the active site serine) (Fig. 2) had been suggested by analogy with nonenzymatic reactions. With rapid reaction techniques, spectrophotometric evidence has been obtained for an additional intermediate before the acyl enzyme in the case of chromophoric substrates. By using first the protein fluorescence emission (Fink and Wildi, 1974)... 

Figure 3. Possible mechanism for (a) formation and (b) breakdown of acyl-enzyme (chymotrypsin) intermediate (3)...

Studies of the ability of the lipase B from Candida antarctica (CAL-B) to catalyse the enantioselective aminolysis of esters by cis- and firms-2-phenylcycloalkanamines (54 n = 1, 3, 4) have been followed up by molecular modelling approaches in order to probe the lipase-catalysed aminolysis mechanism. CAL-B possesses a typical serine-dependent triad, so it was possible, with access to an X-ray crystal structure of CAL-B, to model a series of phosphonamidates (55 n = 1, 3, 4) as analogues of the tetrahedral intermediate (TI) resulting from attack of the amine on the carbonyl of the acyl-enzyme. The results suggested as the most plausible intermediate for the CAL-B-catalysed aminolysis a zwitterionic TI resulting from the direct His-assisted attack of the amine on to a C=0 group of the acyl-enzyme.80... 

Detection of the intermediate is possible if it has a spectrum sufficiently different from that of the enzyme. The cinnamoyl chymotrypsin intermediate is characterised by a UV maximum at 292 nm the acyl papain intermediate JV-benzoylaminothionacetyl papain has a UV maximum at 313 nm. The UV absorptions of the reactions catalysed by papain and chymotrypsin wax and wane in the presence of substrate giving rise to these intermediates. 

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