Tosyl-chymotrypsin

Using this approach, Bizzozero and Zweifel (9) and Bizzozero and Dutler (10) have constructed molecular models of two intermediates (an enzyme-substrate complex and a tetrahedral intermediate) by appropriate modification of the models of stable enzyme-species. The stable enzyme-species used (15, 16) are trypsin-benzamidine complex (TR-B) (17), trypsin-pancreatic trypsin inhibitor complex (TR-PTI) (18, 19) and tosyl-chymotrypsin (Tos-CHT) (20) which are related to enzyme substrate complex, tetrahedral intermediate and acyl-enzyme respectively. 

Sulfonyl fluorides have so far been reported to react at only two amino acid residues, cysteine and serine. The chemical evidence for the reaction of phenylmethylsulfonyl fluoride at the reactive cysteine of papain is indirect (Whitaker and Perez-Villasenor 1968). However, the strong chemical evidence for the attack of phenylmethane sulfonyl fluoride at the serine of chymotrypsin (Gold and Fahrney 1964) has been confirmed by the X-ray crystallographic structure of tosyl-chymotrypsin (Matthews et al. 1967) which had been prepared from tosyl fluoride (Sigler et al. 1966). 

Birktoft J J and D M Blow 1972. The structure of Crystalline Alpha-Chymotrypsin V. The Atomic Structure of Tosyl-Alpha-Qiymotrypsin at 2 Angstroms Resolution. Journal of Molecular Biology 68 187-240. 

Matthews, B.W., Sigler, P.B., Henderson, R., Blow, D.M. Three-dimensional structure of tosyl-a-chymotrypsin. Nature 214 652-656, 1967. 

Tosyl-L-phenylalanine chloromethyl ketone (TPCK) specifically inhibits chymotrypsin by covalently labeling His ". 

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... 

In a subsequent study, the Co coordinated assembly was grafted onto a cross-linked poly(glycidoxypropylmethacrylate) (GMA)-ethylene glycol dimethacrylate (EDMA) support surface. The template and Co ions were then leached out from the matrix. This surface grafted polymer exhibited turnover, Michaelis-Menten kinetics and mechanisms such as inhibition by tosyl phenyl alanyl chloromethyl ketone and inhibition on acetylation of hydroxyl group of 2-hydroxyethylmethacry-late (HEMA), similar to native chymotrypsin. 

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

Circiunstantial support for this mechanism was supplied by the fact that A-tosyl-Phe-CMK, a specific inhibitor of chymotrypsin, did not react with anhydrochymotrypsin [104]. Although both X-ray crystallographic and NMR studies supported the alkylated hemiketal as the structure of the inhibited enzyme, those studies did not prove whether alkylation or hemiketal formation oecurred first [105, 98]. Carbon-13 NMR studies were also used to determine the pKa (7.88-8.1) of the hemiketal hydroxyl and this finding provided the first evidence that serine proteinases could stabilize the ionized form of the alkylated hemiketal, via hydrogen bonds in the oxyanion hole [106,107]. A series of more recent papers has confirmed that hemiketal formation precedes the alkylation step and has shown that the initial, reversible part of the interaction is made up of two discrete stages (a) formation of a Michaelis complex, followed by (b) hemiketal formation [102, 108]. The requirement of an intermediate hemiketal may mean that chloromethyl ketone (CMK) inhibitors should be considered as transition-state [109] analogue inhibitors (see diseussion in seetion on Aldehydes). 

The striking successes achieved by Shaw (1970a) and his coworkers with haloketone derivatives of N-tosyl-phenylalanine and a-N-tosyl-lysine as affinity labels for chymotrypsin and trypsin, respectively, have stimulated their use in a large number of affinity labels. Haloketones are potentially reactive with all the nucleophilic amino acid residues in proteins. Examples of residues modified by haloketones include methionine (Sigman et al. 1970), glutamate (Visser et al. 1971), cysteine (Porter et al. 1971), histidine (Schoellman and Shaw 1963) and serine (Schroeder and Shaw 1971). 

The derivatives of histidine formed by haloketones are probably generally stable to acid hydrolysis. At the very least, histidine is not regenerated following hydrolysis in 6 N HCl for 20 hr. Stevenson and Smillie (1965) in their studies of the modification of chymotrypsin by the chloromethyl ketone of N-tosyl phenylalanine or compound II have shown that oxidation by vapors of performic acid for 2 hr of peptides derivatized by the above reagents yields 3-carboxymethyl... 

Figure 2. Molecular (a), van der Waals (b), and extra radius (c) surfaces of chymotrypsin-tosyl inhibitor complex. The surface is color-coded by hydrophobicity as described in the text red = hydrophobic, blue = hydrophilic, neutral = yellow. The tosyl group is covalently attached to the sidechain hydroxyl of Ser-195. The catalytic triad of His-57, Asp-102, and Ser-195 is shown in green. (The coordinates for this and all other molecules in the following figures are from the Brookhaven Protein Data Bank (14) except where otherwise noted.)... 

It is irreversibly inhibited by 1-3 dibromoacetone, M-(4-dimethy 1 amino-3-5dinitrophenyl)maleimide, A/-ethylmaleimide, and iodoacetic acid. The chymotrypsin inhibitors W-tosyl-L-phenylalanyl-chloromethane (TPCK) and W-tosyl-L-lysylchloromethane (TLCK) alkylate the essential SH group [38]. Di-isopropylfluorophosphate (DFP) alkylates the Tyr residues of bromelain but does not react with its catalytic SH. It causes no inhibition of the caseinolytic activity, but the catalytic efficiency against BAEE is enhanced [75]. 

A) Tosyl-L-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. (B) TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue. 

Histidine 57 is another critical amino acid residue in chymotrypsin. Chemical labeling again provides the evidence for involvement of this residue in the activity of chymotrypsin. In this case, the reagent used to label the critical amino acid residue is A -tosylamido-L-phenylethyl chloromethyl ketone (TPCK), also called tosyl-L-phenylalanine chloromethyl ketone. The phenylalanine moiety is bound to the enzyme because of the specificity for aromatic... 

Structure of N-tosylamido-L-phenylethyl chloromethyl ketone (TPCK), a labeling reagent for chymotrypsin [R represents a tosyl (toluenesulfonyl) group]... 

Tripeptide Ser-His-Asp had been introduced to the CD for simulating the chymotrypsin by the following method. Mix 2-amino ethyl mercaptan salts, 6-0-tosyl-/3-CD and NH4HCO3 with DMF/H2O (1 3) solution under nitrogen protection at 60 C for 4h. After vacuum concentration, separate the product by Sephadex G-10 column. The yield is about 43%. Mix the above product with Boc-Ser (Ot-Bu) -His-Asp (Ot-Bu) OH, dicyclohexyl carbodiimide, 1-hydroxy triazole in DMF solution to obtain 6-[Boc-Ser (Ot-Bu) -His-Asp (Ot-Bu) NHCH2CH2S]-/3-CD. Then treat with the TFA/CH2C12(9 1) mixed solution, vacuum concentrate and freeze-dry. This modified CD can be used as an analogue enzyme to hydrolyze nitrophenol esters. 

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