Complexes acyl-enzyme

The detailed mechanism of inhibition of TEM-2 (class A) enzyme with clavulanate has been established (Scheme 1) [23,24], The inhibition is a consequence of the instability of the acyl enzyme formed between the /1-lactam of clavulanate and the active site Ser-70 of the enzyme. In competition with deacylation, the clavulanate acyl-enzyme complex A undergoes an intramolecular fragmentation. This fragmentation initially provides the new acyl enzyme species B, which is at once capable of further reaction, including tautomeriza-tion to an entity C that is much less chemically reactive to deacylation. This species C then undergoes decarboxylation to give another key intermediate enamine D, which is in equilibrium with imine E. The imine E either forms stable cross-linked vinyl ether F, by interacting with Ser-130 or is converted to the hydrated aldehyde G to complete the inactivation. 

The discovery of the ethylidenecarbapenems, the asparenomycins, as naturally occurring /J-lactamase inactivators in the early 1980s was another striking point in /J-lactamase inhibitor research. The substituted exomethylene function in asparenomycins is a distinctive feature of this class of compounds, which many scientists recognized could be a key factor for /J-lactamase inhibition. The exo cyclic methylene is expected to increase the acylation ability, and form an a,/J-unsaturated ester of the active site serine residue as an acyl-enzyme complex. This ester will be similar in structure to the acyl-enzymes formed from clavulanate and sulfone fragmentation, and will be quite resistant to hydrolytic deacylation. Thus, the exocyclic methylene promotes acylation by the enzyme and subsequently represses deacylation. Based on... 

In contrast, with penicillins, cephalosporins, and monobactams where the substituents are cis to each other across the C3 - C4 bond, clockwise rotation can occur without conflict with protein side chains, and will leave the path open for the water molecule to attack and hydrolyze the ester group in B (Scheme 10). Thus, czs-substituted monobactam, as well as penicillins and cephalosporins are rapidly hydrolyzed by class C enzymes (Scheme 10). If this rotation could be prevented by a suitable structural modification, the access of the water molecule to the ester bond will be blocked, which would result in increased stability of the acyl-enzyme complex. 

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. 

Fig. 5.4. Inactivation of /3-lactamases by cephalosporins (Fig. 5.1, Pathway b). The mechanism of this inactivation is similar to that of class-II inhibitors (Fig. 5.3, Pathway b) and is based on the slow hydrolysis of the acyl-enzyme complex (Pathway b). The normal deacylation of the acyl-enzyme complex represented by Pathway a results in the lost of antibacterial activity of the drug. The ratio between Pathways a and b is determined by the nature of the...

The arrangement of S965, H746, and the oxyanion hole suggests that the classical steps of peptide-bond hydrolysis follow the sequence of the trypsin-like serine proteases, namely the formation of the tetrahedral adduct, the acyl-enzyme complex, and hydrolysis. Tricorn has been shown to exhibit both tryptic and chymotryp-tic specificities (Tamura et al. 1996a). The X-ray structure reveals that specificity for basic PI residues is conferred by D936 which is provided by the diad-related subunit (see Figures 10.9 and 10.10). 

The active site of lipases is characterized by a triad composed of serine, histidine, and aspartate, and acyl-enzyme complexes are the crucial intermediates in all lipase-catalyzed reactions 117,118) (Fig. 13). 

Extensive studies of enzyme-substrate complexes by resonance Raman spectroscopy (RR) have prompted the synthesis of new peptide bond modifications such as thionoesters and dithioesters (Scheme l7)t82-83l within simple model substrates. The resulting acyl-enzyme complexes are especially amenable to RR analysis with cysteine proteases such as papain due to formation of the transient dithioester intermediates. 

Both alkaline proteases form an intermediate, the acyl-enzyme complex, on the reaction coordinate from the amino acid component to the dipeptide, which is formed by the triad Ser-(or Cys-)-His-Asp (or -Glu) (see Chapter 9, Section 9.5). The acyl-enzyme complex can be formed with the help of an activated amino acid component such as an amino acid ester. The complex can react either with water to the undesired hydrolysis product, the free amino acid, or with the amine of the nucleophile, such as an amino acid ester or amide, to the desired dipeptide. The particular advantage of enzyme-catalyzed peptide synthesis rests in the biocatalyst specificity with respect to particular amino acids in electrophile and nucleophile positions. Figure 7.26 illustrates the principle of kinetically and thermodynamically controlled peptide synthesis while Table 7.3 elucidates the specificity of some common proteases. 

Methylene penems constitute good /3-lactamase inhibitors as their hydrolytic metabolism leads to the formation of 1,4-thiazepines, strengthening the thus-formed acyl-enzyme complex link (see Sections 2.03.6.9 and 2.03.12.4). However, compared to penams, penem sulfones are not as good /3-lactamase inhibitors as their half-lives of hydrolysis are too short, making them labile under physiological conditions <1997BML2217>. 

The products arising from cleavage of the azetidinone ring of bioactive penems are of particular interest in the comprehension of an acyl-enzyme complex formation with the target enzymes (see Section 2.03.6.2). 

The electrospray ionization mass spectrometry (ESI-MS) analysis of the incubation between porcine pancreatic elastase (PPE) and the tert-butylammonium salt of clavulanic acid 54 (R1 = R2 = H) at time points of between 3 min and 5h revealed that there were no mass increments relative to PPE. However, when the benzyl derivative 54 (R1 = Bn, R2 = H) was used, a peak was observed at 26187 Da after 3 min, which corresponded to the formation of an initial acyl-enzyme complex. The intensity of the peak decreased significantly and two clear additional peaks at 25 968 and 25 967 Da appeared after 5 min, which corresponded to adducts with mass increments at 77 and 88 Da, respectively. The intensities of both peaks decreased after 60 min and almost disappeared after 5 h. The corresponding />-nitrobenzyl ester 54 (R1 = CH2PhN02, Rz = H) showed similar results except that the formation of adducts appeared slightly faster <2000T5729>. 

The trypsin family of serine proteases includes over 80 well-characterized enzymes having a minimum sequence homology of >21%. Two amino acid residues are absolutely conserved (Cysl82, Glyl96) within their active sites [26,27]. These proteases have similar catalytic mechanisms that lead to hydrolysis of ester and amide bonds. This occurs via an acyl transfer mechanism that utilizes proton donation by histidine to the newly formed alcohol or amine group, dissociation and formation of a covalent acyl-enzyme complex. 

The mechanism of inhibition by benzoxazinones Figure 2.9) is believed to be similar [196, 197] to that of the alternate-substrate isocoumarins, and formation of a covalent acyl-enzyme complex with PPE has been confirmed by X-ray crystallographic studies [198], However, when is a hydrogen atom, deacylation of the acyl-enzyme via intramolecular ring closure can either reform the starting benzoxazinone (O attack) or lead to an isomeric quinazolinedione (N attack). It was shown for HLE and a-chymotrypsin that formation of the quinazolindione occurs faster than normal hydrolysis to the anthranilic acid. 

Many clinically important yff-lactamases are serine proteases that catalyse y5-lactam hydrolysis by a double displacement mechanism involving a covalent acyl-enzyme intermediate. Inhibitors of these enzymes exert their effect by the formation of a stable acyl-enzyme complex. In most cases, this is as a result of changes that take place in the acyl residue after interaction with the enzyme, that is, the inhibitors are mechanism-based. In other cases, the inhibition of yS-lactamases may merely be due to the formation of a relatively stable covalent acyl-enzyme complex without additional alteration [31]. 

The carbapenems are mechanism-based inhibitors which involve acylation of the active-site residue and subsequent rearrangement to a more stable acyl-enzyme species. Knowles and co-workers [32, 33] have demonstrated that the progressive inhibition of the TEM S-lactamase by the olivanic acids is due to the rearrangement of the J -pyrroline intermediate (15) to the tautomeric and thermodynamically more stable zl -pyrroline (16) Scheme 6.3). The resultant acyl-enzyme complex is believed to be stable to subsequent hydrolytic breakdown, thereby disrupting the catalytic activity of the enzyme. 

Clavulanic acid is also a mechanism-based inhibitor and its mode of action is believed to involve ring opening of the initially formed acyl-enzyme complex (18) to the keto-derivative (19), which may then tautomerise to the hydrolytically more stable -amino-acrylate (20) Scheme 6.4). This transiently inhibited form may hydrolyse to re-release active enzyme or react further with the enzyme to produce irreversibly inhibited forms. It has been shown that approximately 115 molecules of clavulanic acid are destroyed per molecule of enzyme before the j8-lactamase is irreversibly inactivated. Whilst irreversibly inactivated forms are known to exist, the nature of these products is not yet known. Possible structures are (21) and... 

AChE attacks the e.ster. substrate through a serine hydroxyl. forming a covalent acyl-enzyme complex. The serine is activated as a nucleophile by the glutamic acid and histidine residues that serve as the proton sink to attack the carbonyl carbon of A(Ti. Choline is released, leaving the acetylat serine residue on the enz.yme. The acetyl-enz.yme intermedi-... 

In these conditions, kinetics of turnover of N-acetyl-L-tryptophan (NATP) with a-chymotrypsin in 65% DMSO at —40°C, show that the half-life of the acyl-enzyme is about 10 hours. When the substrate concentration is greater than the enzyme concentration, all the enzyme is trapped as acyl-enzyme complex at —40°C, and it is therefore possible to isolate it by gel-filtration chromatography on a Sephadex LH-20 column equilibrated in 65% DMSO, pH 5.5 buffer at —40°C. A large amount of NATP (100 of 0.1 M) was added to the... 

In the case of penicillin G, the outward rotation about the C3-C4 bond can occur vithout conflict with protein side chains. It relaxes the strained conformation along this bond previously enforced by the four-membered ring. Moreover, this rotation improves hydrophobic contact of the dimethyl and the sulfur part of the thiazolidine ring of penicillin with two leucine side chains, thus offering an additional driving force for the outward rotation. Experimental evidence is provided by the observed rotation of 35.2° in this direction about the C3-C4 bond that occurs in the acyl-enzyme complex formed between benzyl penicillin and a mutant class A beta-lactamase (RTEM-1) blocked in deacylation [9]. 

Inspection of class A beta-lactamase crystal structures indicated that a water molecule in an equivalent position would be much less activated because there is a serine residue replacing the tyrosine of class C enzymes [7,11]. Attack in a class A enzyme occurs from the opposite side of the ester, with activation of the water molecule occurring through an extension of the hydrogen bond network that is not present in class C enzymes. Hence, rotation about C3-C4 is less critical to the mechanism of deacylation in these enzymes and restricting the rotation should have less impact on the stability of the acyl-enzyme complex. 

The stability of the acyl-enzyme complexes formed with C freundii class C beta-lactamase allowed solution of their structures by X-ray crystallography (Fig. 8). No significant changes in protein structure occurred, except that the side chain of Aspl23 moved to accommodate... 

Optimization of these interactions described above through modification of the acyl side chain increased the affinity over lO -fold, from a value > 10 mM (when none of the interactions are satisfied) to a Km value of 120 nM (for Ro-46-7649 9). The stability of the acyl-enzyme complex also increased (Table 1), possibly because tighter binding of the inhibitor decreases the flexibility of the acyl-enzyme complex and thus the rate of occasional access of water to the ester. 

Finally, the enzymatic nature of CPIA-cholesterol ester formation will be briefly mentioned. None of the enzyme preparations of three known biosynthetic pathways for cholesterol esters, namely, acyl-CoA cholesterol Q-acyltransferase (ACAT), lecithin cholesterol 0-acyltransferase (LCAT), nor cholesterol esterase, was effective in producing CPIA-cholesterol ester from the Ba isomer or CPIA. In contrast, the 9,000 g supernatant or microsomal fractions from liver or kidney homogenate were found to be capable of producing CPIA-cholesterol ester without the addition of any cofactors. As substrate, only the Ba isomer was effective, and none of the 3 other fenvalerate isomers nor free CPIA was effective. The hepatic enzyme preparation also catalyzed hydrolysis of fenvalerate, and in this case all the 4 isomers were utilized as substrates. These facts imply that CPIA-cholesterol ester is formed from the Ba isomer through a transesterification reaction via intermediary acyl-enzyme complex. 

Irreversible inhibitors are effectively esteratic site inhibitors which, like true substrates, react with the hydroxyl group of serine at the catalytic active site. Such inhibitors, sometimes referred to as acid-transferring inhibitors, include the organophosphates, the organo-sulfonates, and the carbamates. All form acyl-enzyme complexes which, unlike substrate-enzyme intermediates, are relatively stable to hydrolysis. Indeed, the phosphorylated enzyme intermediates have half-lives from a few hours to several days (A12), whereas the sulfonated or carbamylated enzyme complexes have much shorter half-lives—several minutes to a few hours. Several strong lines of direct evidence point to the formation of an acyl complex—the isolation of phosphorylated serine from hydrolysates of horse cholinesterase (J2), complex formation and carbamylation (02), and the sulfonation of butyrylcholinesterase by methanesulfonyl fluoride in the presence of tubocurarine and eserine (P6). 

Figure 1 shows the influence of the solution pH on the yield and initial rate of synthesis of CBZ-Lys-Gly-OMe at a temperature of 25°C and [CBZ-Lys] = [Gly-OMe] = 20 mM. The maximum yield is achieved for pH values around 6-6.5. Under thermodynamically controlled conditions, the peptide synthesis occurs between the non-ionic forms of the acyl-donor (CBZ-Lys) and the nucleophile (Gly-OMe). The concentration of these nonionic forms depends on the pH, since an intermediate value between both pK (pHopt = V IpKa +pKb]) is needed in order to achieve high synthetic yields. On the other hand, the reaction rate increases up to pH 7, which is in agreement with the results obtained in the synthesis of the peptide benzoylarginine-leucinamide catalyzed by immobilized trypsin (10), where the authors suggest the nucleophihc attack of the non-ionic form of the nucleophile on the acyl-enzyme complex as the controlling step of the peptide reaction. 

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