Covalent catalytic mechanism

During the catalysis, the enzyme first combines with the substrate to form a kind of mid-complex, which is a covalent mid-product due to some group of enzymes attacking some special group of substrates. According to the different groups of enzymes attacking the substrates, covalent catalysis could be divided into nucleophilic catalysis and electrophilic catalysis. 

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Fig. 2 (A) Antibody 43C9. elicited against a phosphonamidate hapten, catalyzes the hydrolysis of an activated amide. (B) Evidence reveals that antibody 43C9 employs a covalent catalytic mechanism. HisL91 is the nucleophile that attacks the substrate s amide carbonyl. Residue HisH35 assists in transition-state stabilization, while Trj L36 and TryH95 are probably involved in proton transfer.

Catalytic mechanisms employed by enzymes include the introduction of strain, approximation of reactants, acid-base catalysis, and covalent catalysis. 

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

More recently, Kaiser and coworkers reported enantiomeric specificity in the reaction of cyclohexaamylose with 3-carboxy-2,2,5,5-tetramethyl-pyrrolidin-l-oxy m-nitrophenyl ester (1), a spin label useful for identifying enzyme-substrate interactions (Flohr et al., 1971). In this case, the catalytic mechanism is identical to the scheme derived for the reactions of the cycloamyloses with phenyl acetates. In fact, the covalent intermediate, an acyl-cyclohexaamylose, was isolated. Maximal rate constants for appearance of m-nitrophenol at pH 8.62 (fc2), rate constants for hydrolysis of the covalent intermediate (fc3), and substrate binding constants (Kd) for the two enantiomers are presented in Table VIII. Significantly, specificity appears in the rates of acylation (fc2) rather than in either the strength of binding or the rate of deacylation. 

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). 

Whereas standard proteases use serine, cysteine, aspartate, or metals to cleave peptide bonds, the proteasome employs an unusual catalytic mechanism. N-terminal threonine residues are generated by self-removal of short peptide extensions from the active yS-subunits and act as nucleophiles during peptide-bond hydrolysis [23]. Given its unusual catalytic mechanism, it is not surprising that there are highly specific inhibitors of the proteasome. The fungal metabolite lactacystin and the bacterial product epoxomicin covalently modify the active-site threonines and in-... 

The X-ray structure of 33F12 revealed that the catalytic mechanisms of this antibody is significantly dependent on LysH93, which initiates catalysis hy forming a stable covalent conjugated enamine with the ketone substrate that becomes the aldol donor. 

Figure 3.3 (a) Covalent catalysis the catalytic mechanism of a serine protease. The enzyme acetylcholinesterase is chosen to illustrate the mechanism because it is an important enzyme in the nervous system. Catalysis occurs in three stages (i) binding of acetyl choline (ii) release of choline (iii) hydrolysis of acetyl group from the enzyme to produce acetate, (b) Mechanism of inhibition of serine proteases by diisopropylfluorophosphonate. See text for details. 

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]...

Suicide Enzyme Inhibitors. Snicide substrates are irreversible enzyme inhibitors that bind covalently. The reactive anchoring group is catalytically activated by the enzyme itself through the enzyme-inhibitor complex. The enzyme thus produces its own inhibitor from an originally inactive compound, and is perceived to commit suicide. To design a substrate, the catalytic mechanism of the enzyme as well as the nature of the functional gronps at the enzyme active site must be known. Conversely, successful inhibition provides valuable information about the structure and mechanism of an enzyme. Componnds that form carbanions are especially usefnl in this regard. Pyridoxal phosphate-dependent enzymes form such carbanions readily becanse... 

Additional catalytic mechanisms employed by enzymes include general acid-base catalysis, covalent catalysis, and metal ion catalysis. Catalysis often involves transient covalent interactions between the substrate and the enzyme, or group transfers to and from the enzyme, so as to provide a new, lower-energy reaction path. 

As noted earlier, the conversion of benzoylformic acid to benzaldehyde is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme BFD. The proposed catalytic mechanism proceeds through two covalent... 

By virtue of being involved in a catalytic mechanism, a covalently bound intermediate is only transient. In such cases, it has often proved possible to change conditions so that decay... 

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 possible catalytic mechanism of [FeFe] hydrogenases is not well established, since there are fewer redox states accessible to spectroscopy (e.g. by EPR), the proton-accepting base is still being debated, and the protein structure shows a larger variability. Also, the DFT modeling of the reaction cycle is more complicated because the covalently attached cubane [4Fe-4S] subcluster, which seems to play an important role in the electron shuffling, is difficult to include in the calculations. 

Proteases are enzymes catalyzing the hydrolysis of peptide bonds. They form one of the largest enzyme families encoded by the human genome, with more than 500 active members. Based on the different catalytic mechanisms of substrate hydrolysis, these enzymes are divided into four major classes serine/threonine, cysteine, metallo, and aspartic proteases. In serine, cysteine, and threonine proteases, the nucleophile of the catalytic site is a side chain of an amino acid in the protease (covalent catalysis). In metallo and aspartic proteases, the nucleophile is a water molecule activated through the interaction with amino acid side chains in the catalytic site (non-covalent catalysis) (Gerhartz et al., 2002). 

Fig. 3.S Putative catalytic mechanism of the novel active site in fl7. The reaction begins when the nucleophilic oxygen of Thr-1 donates its proton to its own a-amino group and attacks the carbonyl carbon of the substrate. The negatively charged tetrahedral intermediate is stabilized by hydrogen bonding. The acylation step is complete when the a-amino group of Thr donates a proton to the nitrogen of the scissile peptide bond. A covalent bond is formed between the substrate...

Figure 13.12. The thymidylate synthase reaction, and its inhibition by 5-fluoro-deoxyuridinemonophosphate (5-FdUMP). a Overview of the reaction, b The catalytic mechanism. The enzyme forms an intermediate in which it is covalently linked to both the substrate (UMP) and, via the latter, to the coenzyme (bottom center). Resolution of this intermediate does not happen with 5-FdUMP because it requires abstractionof the hydrogen normally found in position 5 of the uracil ring.

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

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