Acyl enzymes elimination

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). 

As for the above cyclopeptides 10, the first step in inactivation process is a selective ring opening. Formation of the acyl-enzyme is then followed by a fast 1,6-elimination and the unmasking of a reactive tethered QIM (Scheme 11.4). 

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

Catalytic site of lipase is known to be a serine-residue and lipase-catalyzed reactions are considered to proceed via an acyl-enzyme intermediate. The mechanism of lipase-catalyzed polymerization of divinyl ester and glycol is proposed as follows (Fig. 3). First, the hydroxy group of the serine residue nucleophilically attacks the acyl-carbon of the divinyl ester monomer to produce an acyl-enzyme intermediate involving elimination of acetaldehyde. The reaction of the intermediate with the glycol produces 1 1 adduct of both... 

Some cephalosporins can be both substrates and inhibitors of /3-lactamases. The acyl-enzyme intermediate can undergo either rapid deacylation (Fig. 5.4, Pathway a) or elimination of the leaving group at the 3 -position to yield a second acyl-enzyme derivative (Fig. 5.4, Pathway b), which hydrolyzes very slowly [35][53], Thus, cephalosporins inactivate /3-lactamases by a mechanism similar to that described above for class-II inhibitors. It has been hypothesized that differences in the rate of deacylation of the acyl-enzyme intermediates derive from their different abilities to form H-bonds. A H-bond to NH in Fig. 5.4, Pathway a, may be necessary to assure a catalytically essential conformation of the enzyme, whereas the presence of a H-bond acceptor in Fig. 5.4, Pathway b, may drive the enzyme to an unproductive conformation. The ratio between hydrolysis and elimination, and, consequently, the relative importance of substrate and inhibitor behaviors of cephalosporins, is determined by the nature of the leaving group at C(3 ). An appropriate substitution at C(3 ) of cephalosporins may, therefore, increase the /3-lactamase inhibitory properties and yield potentially better antibiotics [53]. 

The inhibitory mechanism is shown in figure 16.20. First the transpeptidase (TPase) reacts with one strand of the substrate to form an acyl enzyme intermediate, thus eliminating D-alanine. This intermediate then reacts with another strand to form the cross-link and regenerate the enzyme. Because penicillin is an analog of alanylalanine, it fits... 

Experimental pharmacological investigations into selected acyl enzymes have shown that, besides the depot effect of the modified enzymes, their elimination characteristics changed as did, in some cases, their specific effect, m particular, as for as fibrinolytic enzymes are concerned, acylation could be shown to modify significantly the clearance of these enzymes. The prolonged circulation of... 

The kinetic approach can be more efficiently manipulated than the thermodynamic approach, but serine and cysteine proteases are not perfect acyl transferases. Undesired reactions may take place parallel to acyl transfer, for instance hydrolysis of the acyl-enzyme, secondary hydrolysis of the formed peptide bond, and other undesired proteolytic cleavages of possible protease-labile bonds in reactants and product. The elimination or minimization of these disadvantages can be performed by various manipulations on the level of the reaction medium, the enzyme, and the substrate, as well as on mechanistic features of the process. 

An explanation of these results and determination of the mechanism(s) of inhibition by the isocoumarins required a complex series of kinetic analyses and X-ray crystallographic studies [186]. These studies showed that the mechanistic pathway (see Figure 2.8) was pH-dependent [187] and that different forms of the inhibited enzymes, illustrated by (8a), (8c) and (8d), could be isolated. Ring-opening results in formation of an intermediate acyl-enzyme (8a), which, in some cases, can be isolated but which can also eliminate chloride to produce a reactive quinone imine methide (8b). This reactive intermediate is either trapped by solute or solvent, to produce a second acyl-enzyme (8c) [188] or alkylated by His-57 to produce an irreversibly inactivated enzyme (8d) [189]. The ratio between (8c) and (8d) has been shown to vary widely. 

The enzymatically inactive acyl-urokinase was reactivated in plasma at 37°C with a reactivation half-time of 8 minutes (benzoyl-urokinase) or 10 hours (p-guanidinobenzoyl-urokinase). Upon administration to rabbits, urokinase was more rapidly eliminated than either acyl enzyme (Fig. 11). The results suggest that urokinase is eliminated via the binding to plasma inhibitors. Thus, it could be shown that the clearance of urokinase is modified significantly on acylation. 

On the other hand, nitrilases operate by a completely different mechanism (Scheme 2.101). They possess neither coordinated metal atoms, nor cofactors, but act through an essential nucleophilic sulfhydryl residue of a cysteine [641, 642], which is encoded in the nitrilase-sequence motif Glu-Lys-Cys [643]. The mechanism of nitrilases is similar to general base-catalyzed nitrile hydrolysis Nucleophihc attack by the sulfhydryl residue on the nitrile carbon atom forms an enzyme-bound thioimidate intermediate, which is hydrated to give a tetrahedral intermediate. After the elimination of ammonia, an acyl-enzyme intermediate is formed, which (like in serine hydrolases) is hydrolyzed to yield a carboxyhc acid [644]. 

Hydrolysis of Enol Esters. Enzyme-mediated enantioface-differentiating hydrolysis of enol esters is an original method for generating optically active a-substituted ketones (84—86). If the protonation of a double bond occurs from one side with the simultaneous elimination of the acyl group (Fig. 3), then the optically active ketone should be produced. Indeed, the incubation of l-acetoxy-2-methylcyclohexene [1196-73-2] (68) with Pichia... 

A most significant advance in the alkyne hydration area during the past decade has been the development of Ru(n) catalyst systems that have enabled the anti-Markovnikov hydration of terminal alkynes (entries 6 and 7). These reactions involve the addition of water to the a-carbon of a ruthenium vinylidene complex, followed by reductive elimination of the resulting hydridoruthenium acyl intermediate (path C).392-395 While the use of GpRuGl(dppm) in aqueous dioxane (entry 6)393-396 and an indenylruthenium catalyst in an aqueous medium including surfactants has proved to be effective (entry 7),397 an Ru(n)/P,N-ligand system (entry 8) has recently been reported that displays enzyme-like rate acceleration (>2.4 x 1011) (dppm = bis(diphenylphosphino)methane).398... 

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