Covalent acylation

A steady-state kinetics study for Hod was pursued to establish the substrate binding pattern and product release, using lH-3-hydroxy-4-oxoquinoline as aromatic substrate. The reaction proceeds via a ternary complex, by an ordered-bi-bi-mechanism, in which the first to bind is the aromatic substrate then the 02 molecule, and the first to leave the enzyme-product complex is CO [359], Another related finding concerns that substrate anaerobically bound to the enzyme Qdo can easily be washed off by ultra-filtration [360] and so, the formation of a covalent acyl-enzyme intermediate seems unlikely in the... 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109. It does not catalyze a direct attack of water on the peptide bond instead, a transient covalent acyl-enzyme intermediate is formed. The reaction thus has two distinct phases. In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme. In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated enzyme regenerated. 

The first evidence for a covalent acyl-enzyme intermediate came from a classic application of pre-steady state kinetics. In addition to its action on polypeptides,... 

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... 

Covalent intermediates in serine protease catalysis have played a significant and historical role in our understanding of enzyme mechanism. During catalysis the Substrate will pass through at least two covalent intermediates. The- tetrahedral adduct is formed before generation of the acyl enzyme. Trapping of covalent acyl enzymes from specific substrates is very difficult since in this substrate type the... 

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. 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

This ambiguity was resolved in the experiment shown in Table XL The peptide in this case represents the C-terminal sequence of the B-chain of insulin. The major products were the penta-peptide lacking the N-terminal phenylalanine and free phenylalanine indicating that hydrolysis was the major reaction. In addition, however, there was a significant amount of Phe-Phe. This dipeptide could only come from a transpeptidation involving an acyl transfer. The acyl transfer presumably proceeds via a covalent acyl intermediate. The evidence for this comes from the experiment shown in Table XII where Leu-Tyr-Leu was incubated with both porcine pepsin and penicillopepsin in the presence of a lO-fold excess of C-leucine over Leu-Tyr-Leu. The products, leucine and leucylleucine, were separated by high voltage electrophoresis and analyzed for their specific radioactivity. At most, only traces of radio-... 

The acylium ion is understandably more reactive than a covalent acyl species, and is able to acylate even relatively unreactive secondary and tertiary steroid alcohols. In some situations, however, the acidic reagent causes elimination reactions, with or without rearrangement, so that esterification is inefficient or impossible (p. 105 and 271). 

Figure 1 Diagram of a protease active site. A protease cieaves a peptide at the scissiie bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminai side of the scissiie bond and their prime-side residues C-terminal to the scissiie bond. The cataiytic residues determine the ciass of protease. Serine, cysteine, and threonine proteases hydroiyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, giutamic and metaiioproteases activate a water moiecuie to hydroiyze the peptide bond in a non-covalent manner.

Penicillin inhibits the cross-linking transpeptidase by the Trojan horse stratagem. The transpeptidase normally forms an acyl intermediate with the penultimate d-alanine residue of the d-Ala-d-Ala peptide (Figure 8.29). This covalent acyl-enzyme intermediate then reacts with the amino group of the terminal glycine in another peptide to form the cross-link. Penicillin is welcomed into the active site of the transpeptidase because it mimics the d-Ala-d-Ala moiety of the normal substrate (Figure 8.30). Bound penicillin then forms a covalent bond with a serine residue at the active site of the enzyme. This penicilloyl-enzyme does not react further. Hence, the transpeptidase is irreversibly inhibited and cell-wall synthesis cannot take place. 

In contrast to the equilibrium-controlled approach which ends with a true equUibrium, in the protease-catalyzed kinetically controlled synthesisf l the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. This approach requires the use of acyl donor esters as carboxy components (Ac-X) and is limited to proteases which rapidly form an acyl-enzyme intermediate (Ac-E). Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophihc amino components via an acyl-enzyme intermediate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester, Ac-X, to form a covalent acyl-enzyme intermediate, Ac-E, that reacts, in competition with water, with the amino acid or peptide-derived nucleophile HN to form a new peptide bond (Scheme 3). The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control, and if k4[HN] k3[H20], the peptide product Ac-N should accumulate. However, the soluble peptide product will be degraded if the reaction is not terminated after the acyl donor ester is consumed. 

Methanococcus voltae contains a membrane-bound vanadate-sensitive ATPase [48] that is inhibited by diethylstilbestrol, an inhibitor of eukaryotic P-type ATPases. The purified enzyme is composed of a single subunit (Mr 74 000), forms a covalent acyl-phosphate enzyme intermediate, and is not inhibited by nitrate or bafilomycin [49]. No such ATPase activity has been reported in other archaea. The presence of a second ATPase in M. voltae has been inferred since membranes react with antiserum prepared against the 3 subunit from the V-type ATPase of S. acidocaldarius [50]. Two peptides are detected whose Mr values (51 000 and 65 000) correspond to the masses for the two laigest subunits of the S. acidocaldarius ATPase [51]. There is evidence that ATP synthesis in the M. voltae enzyme is due to the operation of a sodium-translocating ATPase [50]. The relationship of the putative V-like ATPase to the sodium-translocating ATPase has not been established. 

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]. 

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-... 

This represents direct evidence for the existence of a presumably covalent acyl-enzyme intermediate in the a-chymotrypsin-catalyzed hydrolysis of a specific substrate (Fink, 1973b). 

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