Chymotrypsin, 508 Table

This has been found for many series of ester substrates of chymotrypsin (Table 7.1) since the original study of H. Gutfreund and B. R. Hammond in 1959.19 For weakly activated esters, the value of kCM decreases to below that of fe3 because k2 becomes partly rate-determining (equation 7.1). With amides, kcat is very low and k2 is completely rate-determining. The steady state analysis of kcat in relation to k2 and k3 was presented in Chapter 3 [equation 3.22, where kCit = k2kiHk2 + k3)]. Also given there was the relationship between KM and Ks... 

In the course of sequence analysis studies, chymotryptic digests of several proteins and peptides have been examined in detail. These studies have revealed significant chymotryptic cleavage at many bonds often thought to be resistant to chymotrypsin. Table VI lists the types of bonds in nine... 

Also, around the optimum pH of a-chymotrypsin or pH 8, the rate of the hydrolysis reaction was measured, /c at of 2 is over twice that of a-chymotrypsin (Table I) [10]. 

We have determined the rate of hydrolysis of the endo,endo compound containing intramolecular benzoate ion, 4, and shown it to be equal to 1/18 the rate of deacylation of trans-cinnamoyl-g-chymotrypsin (Table II). Furthermore, we find that an increasing dioxane concentration in the solvent increases the reaction rate as seen before. 

Porcine liver esterase (PLE) gives excellent enantioselectivity with both dimethyl 3-methylglutarate [19013-37-7] (lb) and malonate (2b) diester. It is apparent from Table 1 that the enzyme s selectivity strongly depends on the size of the alkyl group in the 2-position. The hydrolysis of ethyl derivative (2c) gives the S-enantiomer with 75% ee whereas the hydrolysis of heptyl derivative (2d) results in the R-monoester with 90% ee. Chymotrypsin [9004-07-3] (CT) does not discriminate glutarates that have small substituents in the 3-position well. However, when hydroxyl is replaced by the much bulkier benzyl derivative (Ic), enantioselectivity improves significantly. 

TABLE 7.1. Kinetic Parameters for the Hydrolysis of Different Peptides by Elastase and Chymotrypsin... 

The effects of various enzymes on the activity of HPLC fractions that inhibited 3H-PCP binding were investigated. As shown in table 1, pronase (0.5 pg/ml), carboxypeptidase A (0.1 unit/ml), and trypsin (3.0 g/ml ) markedly decreased the potency of 10 n units of PCP-like activity. No significant change in activity was. seen when fractions were incubated with alpha-chymotrypsin. 

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). 

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). 

TABLE 11.1 Inhibition of a-Chymotrypsin and Human Leukocyte Elastase by Phenolic Esters of 6-(Chloromethyl)-2-oxo-2H-l-Benzopyran-3-Carboxylic Acid42... 

TABLE 11.2 Inhibition of Human Leukocyte Elastase, a-Chymotrypsin and Thrombin by 5 -Chloropyrid-3 -yl Derivatives at pH 8.0 and 25°C21... 

A series of enzyme and proteins (met-myoglobin, lysozyme, met-hemoglobin, glucose oxidase, a-chymotrypsin) was also immobilized in a-ZrP by Kumar et al. [134]. Binding constant values clearly confirm the high affinity of the various proteins with the host structure (Table 15.4). 

Fig. 9 Correlation of (A) the second order rate constants (k2 = kcatIKM) and (B) the transition stabilization (pATS) with the hydrophobicity (it) of the substituent of the amino acid residue for the cleavage of /V-acetylamino acid methyl esters by a-chymotrypsin. The open symbols are for the points for two branched residues (valine and isoleucine). Data from Table A6.8.

Table A6.8 Cleavage of /V-acetylamino acid methyl esters by a-chymotrypsin."...

Figure 13.8 Temperature response trace (at optimal Tm solution conditions) of the free energy of unfolding (AGu), calculated from Equation 1. Pgen = pepsinogen, Ctsin = a-chymotrypsin, Ctgen = a-chymostrypsinogen, and papain. Note the pH conditions are listed in parentheses and the thermodynamic parameters used in Equation 1 are from those listed in Table 13.2.

In the late 1950s it was shown that imidazole catalyzes the hydrolyses of />-nitrophenyl acetate (7, 76) and that histidine was at the active site of a-chymotrypsin (2). These findings led Katchalski ei al. (39) to synthesize a number of histidine-containing polymers for evaluation as catalysts. Second-order rate constants were calculated on the basis of the concentration of neutral imidazole, that is, k2 = (A bs — .)/a[IM], where k , is the rate constant in the absence of catalyst and a is the fraction ionized. Some of these rate constants appear in Table I. All of the polymers possess less than... 

TABLE 1. Enzymatic Activity of the ST HPMA Polymer Modified Chymotrypsin in the Cieavage of Z-Gly-Leu-Phe-NAp. 

Hubbard and Kirsch (1972) have recently proposed that histidine may act as a nucleophile in a-chymotrypsin acylation reactions of esters having a good leaving group (jO-nitrophenol). This suggestion was based on a similarity in p-value for acylation by p-substituted nitrophenyl and dinitrophenyl benzoates and nucleophilic attack on these compounds by imidazole, in contrast with less positive p-values for hydroxide ion catalysis. Hammett p-values for hydrolysis of substituted phenyl esters are given in Table 6 and show little apparent trend. The values for hydroxide ion and alcoholate ions are... 

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). 

Table III. Determination of Modified Amino Groups of Chymotrypsin...

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). 

Table IV. Effect of DMF Concentration on Synthesis of Z-Tyr-Gly-NH2 by Native and Modifled Chymotrypsin...

Values of a catalytic constant, 27 k computed from (6) are listed in Table V. It is obvious that the imidazole-substituted dodecylpoly-enimines are more than 100 times as effective as the simple imidazole28,29 molecule itself. The catalytic constant for the imidazole-dodecyl polymer in fact approaches that of the enzyme chymotrypsin. 

Immobilization of enzymes to solid supports can increase activity over a wide range of solvents [78]. As seen in Table 3.2, the transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) with 1-propanol by a-chymotrypsin (Scheme 3.2) immobilized to glass is 1-2 orders of magnitude higher than that of the free, lyophilized enzyme. 

Table 3.2 Values of (kc /Km)app (M 1 min-1) x 100 for freely suspended and glass-immobilized a-chymotrypsin in various solvents. In all solvent conditions, the enzyme is 1-2 orders of magnitude more active than the free, lyophilized enzyme [78].

This method was employed for transesterification reactions with both a-chymotrypsin and subtilisin Carlsberg with a variety of H+/Na+ buffers [53]. With both enzymes (which differ widely in secondary and tertiary structures) and two polar solvents, acetonitrile and THF, the activating effect of the solid-state buffer was clearly evident (Table 3.3). The observation that a variety of buffer pairs show success in activating two dissimilar enzymes in synthetically useful solvents makes this method for activation promising and novel. 

Table 3.4 Effect of KCI as a salt matrix on subtilisin Carlsberg and chymotrypsin in anhydrous hexanew [88].

ACE inhibitory peptides have been separated from the skin of skate by Lee et ah (2011). The purified peptides showed IC50 values of 95 and 148 M, respectively, for both peptides isolated from the skin of skate. Further, Lineweaver-Burk plots indicated that the peptides act as noncompetitive inhibitor against ACE. Recently, many inhibitory peptides against ACE are reported (Table 15.2) as natural alternative biofunctional peptides that are safer than that of the existing artificial ACE inhibitory compounds in the market that show some side effects. Various peptides have been isolated from seafood by-products from the fisheries industry such as backbones from tuna (Lee et al., 2010). Tuna backbone has hydrolyzed using various proteases such as alcalase, a-chymotrypsin, neutrase, papain, pepsin, and trypsin to obtain an antioxidant peptide (Je et ah, 2007). 

Hydrolysis of peptides or proteins with acid yields a mixture of free a-amino acids. When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein. Some amino acids may occur only once or not at all in a given type of protein others may occur in large numbers. Table 3-3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of bovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. 

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