Chymotrypsin inactivation

A method for analyzing protein structure based on limited proteolysis. This method is especially useful in investigations of membrane proteins whose membrane association limits the repertoire of techniques that can be gainfully applied to infer structural features. For example, Davis et al used four proteases to assess the topology of yeast H -ATPase reconstituted into phosphatidyl-serine vesicles. Limited proteolysis by trypsin and a-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of... 

Synthesis of Z-Tyr-Gly-NH2 in Aqueous-DMF Solvent Media. Using this modified enzyme, we carried out the synAesis of "Z-Tyr-Gly-NH2", which has never been formed in 100% aqueous system (14), and compart with native chymotrypsin on the effect of organic solvent. To a solution of Z-Tyr-OH (315mg) in Tris buffer (pH 6.7,0.5ml), which contained N,N-dimethylformamide (DW) (0-100%), was added a solution of H-Gly-NH2 HC1 (1 Img) and native or modified chymotrypsin (2mg) in the same Tris buffer. The mixture was incubated at 20°C for 24 hours and heated at lOO C for IS minutes. The products were isolated by HPLC (ODS column, 278nm, 50% acetonitrile). Native chymotrypsin inactivated when concentration of DMF was 50%, while chemically modified chymotrypsin kept its activity even up to 80% (Table IV). 

Measurements of the temperature dependence of enzymatic activity suggested the presence of an intermediate step in enzyme inactivation. The proposed mechanism involves a reversible change to an inactive state, which precedes the final irreversible inactivation step [21-23]. This mechanism could now be refined based on experiments with single enzymes, which detected the intermediate steps directly. This more detailed information allowed the establishment of a tentative model for a-chymotrypsin inactivation (Fig. 25.2d). 

Second, inactivation can be due to a conformational change of the enzyme (214). For example, chymotrypsin inactivation by OH radical, which can be protected by the presence of oxygen, is pH-dependent (212). This pH dependence. 

Besides AT, heparin cofactor II (HCII) is an antic-oagulatory protein enhanced by heparin. HCII inactivates thrombin and the nonclotting enzymes cathepsin-G and chymotrypsin. 

Allaire M, Chernaia MM, Malcolm BA, James MN (1994) Picomaviral 3C cysteine proteinases have a fold similar to chymotrypsin-Kke serine proteinases. Nature 369 72-76 Altman MD, Nalivaika EA, Prabu-Jeyabalan M, Schiffer CA, Tidor B (2008) Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease. Proteins 70 678-694... 

Definition of Ej and E2 eonformations of the a subunit of Na,K-ATPase involves identification of cleavage points in the protein as well as association of cleavage with different rates of inactivation of Na,K-ATPase and K-phosphatase activities [104,105]. In the Ei form of Na,K-ATPase the cleavage patterns of the two serine proteases are clearly distinct. Chymotrypsin cleaves at Leu (C3), Fig. 3A, and both Na,K-ATPase and K-phosphatase are inactivated in a monoexponential pattern [33,106]. Trypsin cleaves the E form rapidly at Lys ° (T2) and more slowly at Arg (T3) to produce the characteristie biphasic pattern of inactivation. Localization of these splits was determined by sequencing N-termini of fragments after isolation on high resolution gel filtration columns [107]. 

The E2 form is not cleaved by chymotrypsin, but trypsin cleaves at Arg (T1) and subsequently at Lys (T2) and tryptic inactivation of E2K or E2P forms is linear and associated with cleavage at Arg" (Ti) [104,108], Inactivation of K-phosphatase is delayed because cleavage of T1 and T2 in sequence is required for inactivation of K-phosphatase activity [105],... 

Baverstock, K., Cundall, RB., Adams, G.E. and Redpath, J.L. (1974). Selective free-radical reactions with proteins and enzymes. The inactivation of a-chymotrypsin, Int. J. Radiat. Biol. 26, 39-44. 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

Bromomethyl-3,4-dibromo-3,4-dihydrocoumarin 1 (Fig. 11.4) and its chloro-methylated analogue 2b rapidly and progressively inactivate a-chymotrypsin and also the activities of a series of trypsin-like proteases. A benzyl substituent characteristic of good substrates of a-chymotrypsin was introduced at the 3-position to make inhibition more selective. This substituted dihydrocoumarin 3 irreversibly inhibited a-chymotrypsin and other proteases. These functionalized six-membered aromatic lactones, and their five- and seven-membered counterparts, 3//-benzofuran-2-ones 2a26 and 4,5-dihydro-3//-benzo[b]oxepin-2-ones 2c,27 were the first efficient suicide inhibitors of serine proteases. Their postulated mechanism of action is shown in Scheme 11.2. 

Coumarincarboxylate derivatives are versatile, efficient, low molecular weight, nonpeptidic protease inhibitors. Both esters and amides behave as time-dependent inhibitors of a-chymotrypsin but the esters are clearly more efficient than the corresponding amides. The criteria for a suicide mechanism are met. The presence of a latent alkylating function at the 6-position (chloromethyl group) is required to produce to inactivation by a suicide mechanism (Scheme 11.3, pathway a). Aryl esters, in particular the meta-substituted phenyl esters are the best inhibitors. Thus, m-chlorophenyl 6-(chloromethyl)-2-oxo-27/-l-benzopyran-3-carboxylate is one of the well-known inactivator of a-chymotrypsin (kJK, = 76(),000M s 1 at pH 7.5 and 25 °C, Table 11.1). 

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

The 6-chloromethyl substituent (series 5 and 6) is required for the inactivation of a-chymotrypsin. Nevertheless, there is only a transient inactivation of HLE and thrombin through the formation of a stable acyl-enzyme in spite of the presence of this group as demonstrated by the spontaneous or hydroxylamine-accelerated reactivation of the treated enzymes (Scheme 11.3, pathway b).21 HLE is specifically inhibited when such an alkylating function is absent (series 7), always through the formation of a transient acyl-enzyme (Table 11.2). 

Bechet, J.-J. Dupaix, A. Blagoeva, I. Inactivation of a-chymotrypsin by new bifunctional reagents halomethylated derivatives of dihydrocoumarins. Biochimie 1977, 59, 231-239. 

Studies on the active agent of the yeast extract have shown it to be nondialyzable, heat labile, and inactivated by trypsin and chymotrypsin as well as silver ions and p-hydroxymercuribenzoate.(7l) These and other properties suggest that the active agent is an enzyme. Sedimentation studies have shown that the yeast photoreactivating enzyme combines with UV-irradiated DNA, in which condition it is more resistant to heat inactivation and inactivation due to silver ions and p-hydroxymercuribenzoate.<75) The... 

Inactivation due to digestive proteases. Therapeutic proteins would represent potential targets for digestive proteases such as pepsin, trypsin and chymotrypsin. 

Figure 7. Two examples of irreversible inactivators that are not suicide substrates a) TPCK, a classic" affinity label of the serine protease chymotrypsin, b) ZFK-CH2-mesitoate, a quiescent" affinity label of the cysteine protease cathepsin B, and c) the kinetic scheme for both forms of affinity label-inactivation.

Chymotrypsin, in addition to its proteolytic activity, can also function as an esterase.1 It is inactivated by D.F.P., etc. (p. 186). The esterases firmly bind the phosphorus of D.F.P., and in the case of chymotrypsin the reaction is bimolecular, yielding a crystalline derivative containing two isopropoxy groups and one atom of phosphorus per protein molecule, but no fluorine.2... 

Increased understanding of reaction mechanisms in the 1940s and 1950s pinpointed general acid or base catalysis as likely to be of importance in many hydrolytic reactions. The imidazole nucleus in histidine was the obvious center in proteins to donate or accept protons at physiological pH. The involvement of histidine was shown by photochemical oxidation in the presence of methylene blue (Weil and Buchert, 1951) which destroyed histidine and tryptophan and inactivated chymotrypsin and trypsin. 

Because PMSE fails to inactivate acetylcholinesterase, this reagent is much less toxic than diisopropylfluoro-phosphate, and is also recommended as an alternative to the neurotoxic fluorophosphates and fluorophospho-nates. PMSE is freshly prepared as a 1-3 mM solution in water (higher concentrations will precipitate spontaneously). A better procedure is to first prepare a 20 mM PMSE solution in 2-propanol or dioxane this solution can then be added to the biological fluid with vortex mixing to achieve a 1-3 mM final concentration as a homogeneous solution. One should confirm that the alcohol or dioxane has little or no undesirable effect on enzymes or proteins of interest. See Chymotrypsin Protease Inhibitor Cocktails ... 

Selected entries from Methods in Enzymology [vol, page(s)] Sulfonylation reaction, 11, 706 reaction kinetics, 11, 707 second-order rate constants for inactivation of chymotrypsin, trypsin, and acetylcholine esterase by PMSE and related sulfonylat-ing agents, 11, 707 reactivation of PMS-chymotrypsin, 11, 710 as inhibitor [of calcium-activated factor, 80, 674 of cathepsin G, 80, 565 of crayfish trypsin, 80, 639 of elastase, 80, 587 of pro-lylcarboxypeptidase, 80, 465 of protease Re, 80, 691 of protease So, 80, 695 of protein C, 80, 329] proteolysis, 76, 7. 

The active-site-directed inhibitor tosylphenylalanine chloromethyl ketone that specifically and irreversibly inhibits chymotrypsin. This chloroketone inhibitor relies on its toluene sulfonyl (or tosyl) group for binding into the aromatic binding pocket of chymotrypsin s active site. Inactivation occurs by alkylation of histidine-57 (pseudo-first order rate constant 0.2 min ). See Chymo-trypsin... 

Multipoint attachment to a support protects the enzyme from inactivation by organic solvents. Mozhaev et al. (1990) have recently demonstrated that covalent linkage to polyacrylamide gel stabilizes df-chymotrypsin from denatmation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. 

Mozhaev, V.V., Sergeeva, M.V. Belora, A.B. and Khmelnitsky, Y.L. (1990) Multipoint attachment to a support protects enzyme from inactivation by organic solvents -chymotrypsin in aqueous solutiuons of alcohols and diols. Biotechnol. Bioeng., 35, 653-659. 

Large differences in sensitivity toward interfacial inactivation were observed between a-chymotrypsin and Candida rugosa lipase [56]. The lipase was most rapidly inactivated by 1-butanol and tolerated the hydrophobic hydrocarbons quite well, while the opposite was true for a-chymotrypsin. A detailed study of interfacial inactivation by 12 different solvents, all having log P values around 4, revealed... 

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