Chymotrypsin enzyme-modification

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

The use of PAL in enzyme modifications was first described in 1962, when Westheimer and coworkers reported on the use of a diazoacetyl group to inactivate chymotrypsin [29]. Considerable research on the development of new PAL reagents has taken place ever since [27, 28, 30-36], but only a few photophores, which largely meet the above-mentioned requirements, are being used nowadays in A/BPs. These are aryl azides (first reported use in 1969) [37], diazirines (1973) [38] and benzophenones (1973) [39]. The chemistry of these three photoreactive groups after photolysis as well as their use in recently reported A/BPs will be discussed in Sects. 3.1-3.5. 

Classical approaches to chemical enzyme modification, however, often suffer from lack of regio-selectivity, which can yield heterogeneous and irreproducible enzyme mixtures. For example, preparation of methyl-chymotrypsin, -subtilisin or -trypsin using methyl sulfonate reagents, originally used to methylate the histidine of the... 

In a chemical enzyme-modification experiment conducted by E. F. Jansen and colleagues in 1949, chymotrypsin was incubated with 32P-Iabeled DFP and then hydrolyzed with a strong acid. Separation of the constituent amino acids revealed 1 mol of labeled Ophosphorylserine per 25,000 g of chymotrypsin. Since DFP is a potent inhibitor of the enzyme chymotrypsin, what might we infer about the amino acid side-chain composition of the active site ... 

Since for chymotrypsin Mr = 25,000, only a single serine had reacted out of a total of 27. This indicated that a particular serine is an important component of the active site. This experiment is the archetypal form of many enzyme-modification procedures that are now used frequently to identify active-site constituents. 

PK Banerjee, GL Amidon. Physicochemical Property modification strategies based on enzyme substrate specificities II Alpha-chymotrypsin hydrolysis of aspirin derivatives. J Pharm Sci 70 1304, 1981. 

HPMA copolymers are water-soluble biocompatible polymers, widely used in anticancer drug delivery (reviewed in Reference [22]). HPMA copolymers containing reactive groups at side-chain termini were previously used for the modification of trypsin [23], chymotrypsin [23,24], and acetylcholinesterase [25]. The modification dramatically increased the acetylcholinesterase survival in the blood stream of mice and the thermostability of modified enzymes when compared to the native proteins. However, the modification involved multipoint attachment of the copolymers to the substrates, which may cause crosslinking. To modify proteins or biomedical surfaces by one point attachment, semitelechelic polymers should be used. 

Chemical modification of proteins has been extensively studied over the years to identify which amino acids are involved in catalysis. Much less work has been carried out on its influence on enzyme stability. Chemical modification of proteins may yield stabilization, destabilization or no effect at all. Martinek and Berezin (1978) reported the dependence of the thermostability of chymotrypsin on the degree of alkylation of its amino groups up to 30% alkylation the stability rose slightly at 90% substitution stability increased markedly, with a maximum (110-fold) at 95% stability fell to nearly initial values when 100% amino groups were modified. (With these modifications, the optimum pH of the errzyme can change and one must therefore be cautious in comparing two different... 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

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. 

In organic solvents, enzymes react at the same active site as in water covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents (Zaks, 1988a). Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin towards competitive inhibitors reverses. 

Efficient modification steps through the proper orientation of the inhibitor reactive group to the enzyme nucleophile is realized by covalent bond formation. A classic example of this type is the modification of a methionine residue of chymotrypsin by /7-nitrophenyl bromoacetyl a-aminoisobutyrate (26)47). In this instance, the reactive group (bromoacetyl) is fixed at the locus near the active site through a covalent bond by means of acyl enzyme intermediates. 

The facile reaction of CAA and BAA with nucleosides and nucleotides is one example of many of the applications of the bifunctional reactivity of halogenated aldehydes and ketones in modification of biomolecules. In an early example of the extensive use of halogenated ketones as protease substrate analogues, l-V-tosylamido-2-phenylethyl chloro-methyl ketone (TPCK) 30 was synthesized as a chymotrypsin substrate analogue. Stoichiometric inhibition was accompanied by loss of one histidine residue as a result of alkylation by the chloromethyl moiety68. A host of similar analogues were subsequently prepared and used as selective enzyme inhibitors, in particular for the identification of amino acid residues located at enzyme active sites69. 

In our initial research on semisynthetic enzymes, we examined briefly the modification of the serine proteinase a-chymotrypsin, perhaps the best understood of the proteolytic enzymes. A logical choice as a residue for alkylation in the active site of a-chymotrypsin is His-57. However, an examination of a three-dimensional model (Lab Quip) of chymotrypsin in which coenzyme analogs were covalently attached to His-57 suggested strongly that such modifications would block completely the enzyme s active site region and that the probability of new reactions being catalyzed by the modified enzyme would be low. Another possible site of modification of chymotrypsin that could be considered was Met-192. This residue, located on the periphery of the... 

Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin is produced in the inactive form, the proenzyme, as chymotrypsinogen. This type of inactive precursor to an enzyme is known as a zymogen. 

A number of proteolytic enzymes participate in the breakdown of proteins in the digestive systems of mammals and other organisms. One such enzyme, chymotrypsin, cleaves peptide bonds selectively on the carboxylterminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine (Figure 91). Chymotry psin is a good example of the use of covalent modification as a catalytic strategy. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate. This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis. 

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