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Chymotrypsin intermediate

There is now convincing evidence that an acyl chymotrypsin intermediate is formed from both specific and non-specific substrates (Bender and Kezdy, 1964 Bender et al., 1964). This intermediate is undoubtedly an acylserine. Acyl- and phosphorylserine derivatives have been isolated and identified. In view of evidence such as a D2 O solvent isotope effect ( h2oAd2o) 2-3 for both acylation and deacylation (Bender and Hamilton, 1962), alcohol and amine nucleophiles showing little dependence on the p/iTa-value of the nucleophile in reaction with furoyl enzyme (Inward and Jencks, 1965), and the influence of increasing steric bulk in the acyl group (Fife and Milstien, 1967 Milstien and Fife, 1968,.1969), consistent... [Pg.32]

Figure 3. Possible mechanism for (a) formation and (b) breakdown of acyl-enzyme (chymotrypsin) intermediate (3)... Figure 3. Possible mechanism for (a) formation and (b) breakdown of acyl-enzyme (chymotrypsin) intermediate (3)...
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. [Pg.319]

Figure 2 Time-resolved ESI-TOF-MS. (a) Diagram with the experimental design, (b) Chymotrypsin reaction with p-nitrophenyl acetate under single-turnover conditions monitored by time-resolved ESI-TOF-MS. The decay of chymotrypsin and formation of the acetyl-chymotrypsin intermediate is observed over a time course of 45 s. (c) Kinetic analysis of chymotrypsin decay and acetyl-chymotrypsin formation. For both traces the rate constant was 0.1 s . ... Figure 2 Time-resolved ESI-TOF-MS. (a) Diagram with the experimental design, (b) Chymotrypsin reaction with p-nitrophenyl acetate under single-turnover conditions monitored by time-resolved ESI-TOF-MS. The decay of chymotrypsin and formation of the acetyl-chymotrypsin intermediate is observed over a time course of 45 s. (c) Kinetic analysis of chymotrypsin decay and acetyl-chymotrypsin formation. For both traces the rate constant was 0.1 s . ...
Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. [Pg.519]

The first intermediate—7r-chymotrypsin—displays chymo-trypsin activity. Suggest proteolytic enzymes that might carry out these cleavage reactions effectively. [Pg.531]

For many serine and cysteine peptidases catalysis first involves formation of a complex known as an acyl intermediate. An essential residue is required to stabilize this intermediate by helping to form the oxyanion hole. In cathepsin B a glutamine performs this role and sometimes a catalytic tetrad (Gin, Cys, His, Asn) is referred too. In chymotrypsin, a glycine is essential for stabilizing the oxyanion hole. [Pg.877]

Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide . Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide .
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. [Pg.63]

The CD-mediated cleavage of p-N02C6H4NHC0CF3 proceeds by acyl transfer to a-CD. Since the trifluoracetyl-CD, so produced, hydrolyses fairly quickly even at pFI7, the overall reaction shows true catalysis. Thus, for the reaction in (27), a-CD behaves as a model enzyme and shows three of the features of chymotrypsin (i) it provides a hydrophobic binding site (ii) it catalyses the loss of leaving group and (iii) the reaction proceeds through an acyl intermediate (Komiyama and Bender, 1977 Bender and Komiyama, 1978). [Pg.46]

In the reaction with PNPA, myristoylhistidine [29] in a cationic micelle rapidly forms acetylimidazole as a fairly stable intermediate which is readily observable at 245 nm. On the other hand, a mixed micelle of [29] and N,N-dimethyl-N-2-hydroxyethylstearylammonium bromide [30] leads to the formation and decay of the intermediate, indicating that the acetyl group is transferred from imidazole to hydroxyl groups (Tagaki et al., 1977 Tagaki et al., 1979). This can be a model of cr-chymotrypsin which catalyses hydrolysis of PNPA (non-specific substrate) by initial acylation of the histidyl imidazole followed by acyl transfer to the seryl hydroxyl group (Kirsh and Hubbard, 1972), as indicated schematically in (12). [Pg.457]

Chymotrypsin catalysis takes place through a three-step process, equation (11), where ES is an enzyme substrate complex which breaks down to give an acylated enzyme intermediate, ES and Pj,... [Pg.30]

A nitrogen isotope effect (1 006,1 010, and 1 006 at pH 6 73, 8 0, and 9 43) has been observed in the chymotrypsin-catalysed hydrolysis of NTacetyl-L-tryptophanamide which requires the C—N bond of the amide to be broken in the rate-determining step (O Leary and Kluetz, 1972). The isotope effect is similar to that observed for the reaction of amides with hydroxide ion which is known to proceed through a tetrahedral intermediate. [Pg.34]

All of this evidence supports the existence of tetrahedral intermediates in a-chymotrypsin-catalysed reactions, but it should be noted that O-exchange with water is not observed in deacylation of cinnamoyl- 0-chymotrypsin, in contrast with the hydrolysis of O-cinnamoyl-N-acetylserinamide where such exchange is detected (Bender and Heck, 1967). Lack of exchange in the enzyme reaction could reflect interactions of the tetrahedral intermediate with the protein. [Pg.34]

Numerous suggestions have been made that enzymes might owe part of their catalytic efficiency to the opportunity they afford for stabilization of intermediates or transition states by hydrogen bonding to functional groups near the active site. For example, in the case of (x-chymotrypsin this might be represented as in [43] where... [Pg.56]

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... [Pg.32]

This endopeptidase [EC 3.4.21.4], a member of the peptidase family SI, hydrolyzes peptide bonds at Arg—Xaa and Lys—Xaa. See Chymotrypsin Catalytic Triad Acyl-Serine Intermediate... [Pg.688]

TETRAHEDRAL INTERMEDIATE ESTER HYDROLYSIS MECHANISM CHYMOTRYPSIN SERPINS (Inhibitory Mechanism)... [Pg.784]

ACYL-SERINE INTERMEDIATE CHYMOTRYPSIN CATALYTIC TRIAD TRYPTOPHANASE TRYPTOPHAN SYNTHASE T state,... [Pg.786]

The tetrahedral intermediate is generated by a nucleophilic attack on the carbonyl carbon atom of the activated nucleophile, which in the case of chymotrypsin and trypsin is the catalyhc Serl95 (Table 4.3). It is important to note that a small structural rearrangement occurs when the planar carbonyl moiety is converted into the tetrahedral intermediate (Scheme 4.4). [Pg.52]

Fig. 2. Model image of a typical substrate bound to ot-Chymotrypsin. (a) Binding of the substrate, (b) Three additional hydrogen bonds stabilize the intermediate oxyanion. Fig. 2. Model image of a typical substrate bound to ot-Chymotrypsin. (a) Binding of the substrate, (b) Three additional hydrogen bonds stabilize the intermediate oxyanion.
Surprisingly few investigations appear to have been made into selective deacylation with the aid of enzymes. a-Chymotrypsin is very sluggish in hydrolyzing acetates of simple nucleosides and nucleotides, but dihydrocinnamic (3-phenylpropanoic) esters appear to be more satisfactory as substrates, and, with such derivatives of nucleosides, it seems that selective deacylation may be achieved enzymi-cally.178 Thus, enzymic hydrolysis of 2 -deoxy-3, 5 -di-0-(dihydrocin-namoyl)uridine gave the 3 -ester as the only intermediate to the... [Pg.43]


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See also in sourсe #XX -- [ Pg.433 ]




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