Chymotrypsin catalytic activity

For many solubilized enzymes the greatest catalytic activity and/or changes in conformation are found at R < 12, namely, when the competition for the water in the system between surfactant head groups and biopolymers is strong. This emphasizes the importance of the hydration water surrounding the biopolymer on its reactivity and conformation [13], It has been reported that enzymes incorporated in the aqueous polar core of the reversed micelles are protected against denaturation and that the distribution of some proteins, such as chymotrypsine, ribonuclease, and cytochrome c, is well described by a Poisson distribution. The protein state and reactivity were found markedly different from those observed in bulk aqueous solution [178,179],... 

RME shows particular promise in the recovery of proteins/enzymes [12-14]. In the past two decades, the potential of RME in the separation of biological macromolecules has been demonstrated [15-20]. RMs have also been used as media for hosting enzymatic reactions [21-23]. Martinek et al. [24] were the first to demonstrate the catalytic activity of a-chymotrypsin in RMs of bis (2-ethyl-hexyl) sodium sulfosuccinate (Aerosol-OT or AOT) in octane. Since then, many enzymes have been solubilized and studied for their activity in RMs. Other important applications of RME include tertiary oil recovery [25], extraction of metals from raw ores [26], and in drug delivery [27]. Application of RMs/mi-croemulsions/surfactant emulsions were recognized as a simple and highly effective method for enzyme immobilization for carrying out several enzymatic transformations [28-31]. Recently, Scheper and coworkers have provided a detailed account on the emulsion immobiUzed enzymes in an exhaustive review [32]. 

We present here the mechanisms for four enzymes chymotrypsin, hexoldnase, enolase, and lysozyme. These examples are not intended to cover all possible classes of enzyme chemistry. They are chosen in part because they are among the best understood enzymes, and in part because they clearly illustrate some general principles outlined in this chapter. The discussion concentrates on selected principles, along with some key experiments that have helped to bring these principles into focus. We use the chymotrypsin example to review some of the conventions used to depict enzyme mechanisms. Much mechanistic detail and experimental evidence is necessarily omitted no one book could completely document the rich experimental history of these enzymes. Also absent from these discussions is the special contribution of coenzymes to the catalytic activity of many enzymes. The function of coenzymes is chemically varied, and we describe each as it is encountered in Part II. 

S-chymotrypsin. The pKa of 6.8 represents the ionization of the catalytically important base at the active site, whereas the high-pH ionization is due to the a-amino group of He-16, which holds the enzyme in a catalytically active conformation. This conformationally important ionization does not affect cat— which usually gives a sigmoid curve of pKa 6 to 7—but it does cause an increase of Km at high pH. 

Schematic diagrams of the amino acid sequences of chymotrypsin, trypsin, and elastase. Each circle represents one amino acid. Amino acid residues that are identical in all three proteins are in solid color. The three proteins are of different lengths but have been aligned to maximize the correspondence of the amino acid sequences. All of the sequences are numbered according to the sequence in chymotrypsin. Long connections between nonadjacent residues represent disulfide bonds. Locations of the catalytically important histidine, aspartate, and serine residues are marked. The links that are cleaved to transform the inactive zymogens to the active enzymes are indicated by parenthesis marks. After chymotrypsinogen is cut between residues 15 and 16 by trypsin and is thus transformed into an active protease, it proceeds to digest itself at the additional sites that are indicated these secondary cuts have only minor effects on the enzymes s catalytic activity. (Illustration copyright by Irving Geis. Reprinted by permission.)...

The amount of water required by chymotrypsin and subhlisin for catalysis in organic solvents is several hundred molecules per protein molecule, less than required to form a monolayer on the surface. While subhlisin and a-chymotrypsin act as catalysts in a variety of dry organic solvents, the vastly different catalytic activities in these organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. 

Catalytic activities of a-chymotrypsin and Subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared (Ke, 1998). Direct suspension of the lyophilized enzyme in the solvent containing 1% water was compared with precipitation of the same enzyme from its aqueous solution by a 100-fold dilution with anhydrous solvent. The reaction rate in a given non-aqueous enzymatic system was found to depend on the nature of both enzyme and solvent, but to depend strongly on the mode of enzyme preparation. 

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

Though a-chymotrypsin has not an extremely high selectivity in binding and catalytic activity compared with other enzymes, it is one of the most well-studied enzymes. 

IM-COOH-OH cooperation. Polymers such as poly(4(5)-vinylimidazole-co-7-vinyl-7-butyrolactone), poly(IM-la), and poly(4(5)-vinylimidazole-co-acrylic acid-covinyl alcohol) derived from poly(4(5)-vinylimidazole-co-methyl acrylate-co-vinyl acetate), both of which contain imidazole, carboxylic acid and hydroxyl moieties are synthesized and studied as a model of a-chymotrypsin (29). The former has a relatively ordered sequence and the latter has a random one. Results are tabulated in Table 11. The polymers cited in the Tabel contain a similarly low quantity of imidazole moiety, so that the cooperation of two subsequent imidazole moieties need not be discussed. Polymers such as L-84, L-68, M-83 and A-84 have higher catalytic activities than the polymer V-82. This suggests that the catalytic activity of the imidazole moiety in the polymers is much promoted by the carboxylate moiety in the polymers. The catalytic activities of L-84 and L-68 which have an ordered sequence are more than twice as high as that of M-83, having a random sequence. From these results it is concluded that the introduction of the hydroxyl moiety which has little cooperative effect on the imidazole moiety in V-82 in this reaction conrfition into imidazole and carboxylate — containing polymer, increases... 

Usually oxime has a high nucleophilicity in dissociated form, but it is useless in a moderate aqueous solution for its high pK value (pK = 11 12). The oxime moiety which is bound to poly(4-vinylpyridine) by the quartemization shows a lower pK value (pK = 8.5 0.3) and high nucleophilicity in the hydrolysis of PNPA(5) (95). The catalytic activity of the polymer(X) reaches a half of that of a-chymotrypsin showing ka = 200 M-1 sec-1 at pH = 8, where m is 12. 

The catalytic activity of artificial chymotrypsin in the hydrolysis of m-tert-butylphenyl acetate (k = 2.8xl02 s 1, KM = 13xl05M) was found to be close to the activity of chymotrypsin in the hydrolysis of p-nitrophenyl acetate (k,.at = l.lxlO2 s 1, KM = 4x105M). Another example of mimicking enzyme catalysis by P-cyclodextrin is general acid-base-catalyzed hydrolysis and nitrosation of amines by alkyl nitrites (Iglesias, 1998). 

Several vital processes rely on clan PA peptidases. Chief among them are blood coagulation and the immune response, which involve cascades of sequential zymogen activation. In both systems, the chymotrypsin-fold peptidase domain is combined with one more associated protein domains, including apple, CUB, EGF, fibronectin, kringle, sushi, and von Willebrand factor domains. These protein domains are on the N-terminus as an extension of the propeptide segment of the peptidase. Such a trend of N-terminal-associated domains in the SIA peptidase family is common across all forms of life. The domain architecture pairs well with the zymogen activation mechanism, which liberates the proper N-terminus to enable catalytic activity. Often, the associated protein domains remain attached to... 

N-bromoacetamide, which is less reactive than NBS at pH 4.0 and consequently requires more time to oxidize tryptophan residues, similar results were obtained (Fig. 27, curve B). Extrapolation of the initial steep portion of the inactivation curve obtained with NBA (Fig. 26) gives a value for tryptophan destruction of about 1 mole per mole of protein. This might suggest the association of one of the seven tryptophan residues present in the chymotrypsin molecule with the maintenance of catalytic activity. A similar involvement of one tryptophan residue in the reaction of diisopro-pylphosphorofluoridate with a-chymotrypsin is suggested by comparison of... 

We report the results from a molecular dynamics simulation of the serine protease y-chymotrypsin (y-CT) in hexane. The active site of chymotrypsin contains the "catalytic triad" which consists of Ser-His-Asp. y-CT suspended in nearly anhydrous solvents has been found to be catalytically active. In order for proteins to retain their activity in anhydrous solvents some water molecules are required to be present. These "essential waters" have been suggested to function as a molecular lubricant for the protein. Hexane, having a dielectric constant of 1.89, is a suitable non-aqueous solvent for enzymatic reactions. The low dielectric constant of hexane allows it to not compete with the protein for the essential water and allows enzymes to retain their catalytic activity. y-CT in hexane is thus an ideal system to further explore the effect of non-aqueous solvation on protein structure, function and dynamics. 

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