A-Chymotrypsin activities

Figurel.10. Stereo diagram of the acyl moiet of the spin-labeled tryptophanyl-acylenzyme reaction intermediate of a-chymotrypsin. Active site residues close to the acyl moiety are labeled (Makinen et al., 1998). Reproduced with permission.

I. Willner, M. Lion-Dagan, S. Rubin, J. Wonner, F. Effenberger, and P. Bauerle, Photoregulation of a-chymotrypsin activity in organic media effects of bioimprinting, Photochem. Photobiol., 59, 491-496 (1994). 

If the comparison of Lewis acid catalysis vs general acid-base catalysis is extended to the specific examples of carboxypeptidase A and a-chymotrypsin, then it is interesting to note that the function of the carboxypeptidase active site catalytic residues (33, 34) appears to involve activation of the substrate for the chemical transformation primarily via Lewis acid catalysis (see Scheme III, Section IV). In contrast, the function of the a-chymotrypsin active site catalytic residues appears to involve the activation of the hydroxyl of Ser-195... 

Cohen, S. G., Vaidya, V. M., and Schultz, R. M., 1970, Active site of a-chymotrypsin activation by association-desolvation, Proc. Natl. Acad. Sci. USA 66 249. 

Sigler, P.B., et al. Structure of crystalline a-chymotrypsin II. A preliminary report including a hypothesis for the activation mechanism. /. Mol. Biol. 35 143-164, 1968. 

Active a-chymotrypsin is produced from chymotrypsinogen, an inactive precursor, as shown in the color figure on page 530. 

All peptidases within a family will have a similar tertiary structure, and it is not uncommon for peptidases in one family to have a similar structure to peptidases in another family, even though there is no significant sequence similarity. Families of peptidases with similar structures and the same order of active site residues are included in the same clan. A clan name consists of two letters, the first representing the catalytic type as before, but with the extra letter P , and the second assigned sequentially. Unlike families, a clan may contain peptidases of more than one catalytic type. So far this has only been seen for peptidases with protein nucleophiles, and these clans are named with an initial P . Only three such clans are known. Clan PA includes peptidases with a chymotrypsin-like fold, which besides serine peptidases such as chymotrypsin... 

The conformations of the locked substrates may be only partially restricted. Indeed, this is the case for many locked substrates. Neither of the substrate analogs under consideration, for example, is restricted to only one conformation. The conformation of 25 which is active with a-chymotrypsin, however, is reasonably well known, whereas the active conformation of 24 is still controversial. 

In conclusion, one must be aware of these limitations on the use of locked substrate analogs. The problems encountered in the study of a-chymotrypsin are perhaps more severe than for most other enzymes, since a-chymotrypsin normally acts on large, polymeric substrates and is relatively nonspecific. The active site of a-chymotrypsin therefore potentially can bind small substrates such as D24 in a variety of ways. Ideally, larger conformationally restricted substrates should give more information about the active site of a-chymotrypsin. However, besides the increased problems involved in synthesizing these larger substrates, there is the problem of increased possibility of uncertainty in their conformations. 

The activity of a-chymotrypsin was found to be insensitive to the R value, i.e., from the size of the reversed micelles. This was taken as an indication that this enzyme is able to create its own micelles in the hydrocarbon rather than occupy empty ones and that the so-called exclusion effect, i.e., protein larger than the empty micelle cannot be solubilized, is incorrect [181,182],... 

Figure 9-6. Selective proteolysis and associated conformational changes form the active site of chymotrypsin, which includes the Aspl 02-His57-Ser195 catalytic triad. Successive proteolysis forms prochymotrypsin (pro-CT), Jt-chymotrypsin (jt-CT),and ultimately a-chymotrypsin (a-CT), an active protease whose three peptides remain associated by covalent inter-chain disulfide bonds.

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. 

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

Dive, G., D. Dehareng, and J. M. Ghuysen. 1994. Detailed Study of a Molecule in a Molecule N-Acetyl-L-tryptophanamide in an Active Site Model of a-Chymotrypsin. J. Am. Chem. Soc. 116, 2548-2556. 

Zoungrana et al. (1997) and Norde and Zoungrana (1998) investigated the influence of adsorption on the structure, structure stability and biological activity of a proteolytic enzyme, a -chymotrypsin. The enzyme was adsorbed from 0.01 M phosphate buffer at pH 7.0 and at 22°C onto solid surfaces of different hydrophobicities and morphologies. 

The addition of the (EO)8 -oligomers to the PS surface resulted in retention of some of the enzymatic activity of adsorbed a -chymotrypsin, whereas this activity was completely lost in the absence of the grafted oligomers. The short (EO)8 chains trapped between the adsorbed protein... 

Fig. 11. Temperature dependency of the specific activity of a-chymotrypsin in solution (o), adsorbed on silica ( ), Teflon (x), polystyrene (A) and polystyrene —(EO)g ( ). Conditions as in Fig. 9. (Redrawn from Zoungrana and Norde...

Szleifer I (1997) Protein adsorption on surfaces with grafted polymers a theoretical approach. Biophys J 72 595-612 Tanford C (1973) The hydrophobic effect. John Wiley Sons, Inc., Hoboken Van Dulm P, Norde W, Lyklema J (1981) Ion participation in protein adsorption at solid surfaces. J Colloid Interf Sci 82 77-82 Zoungrana T, Findenegg GH, Norde W (1997) Structure, stability and activity of adsorbed ensymes. J Colloid Interf Sci 190 437-448 Zoungrana T, Norde W (1997) Thermal stability and enzymatic activity of a-chymotrypsin adsorbed on polystyrene surfaces. Colloid Surf B 9 157-167... 

Esters of higher diacids (e.g., dimethyl glutarates) have also proven quite informative. Thus, dimethyl 3-hydroxyglutarate (7.31) was hydrolyzed by PLE to the monoester of (S)-configuration (7.32), but the ee was low (ca. 20%) [55]. In contrast, a-chymotrypsin yielded the (R)-monoester with ee values of 60-70%, indicating large differences in the active sites of these... 

The enzymatic hydration of lactones is also documented, a variety of hydrolases having demonstrated activity. Very detailed kinetic studies have, for example, been published on the hydrolysis of oxazolones (7.78, R = H or Me, R = Me or aryl, R" = Me or Ph) catalyzed by a-chymotrypsin [163], These compounds are interesting from a chemical point of view, being enolic lac-... 

Steitz TA, Henderson R, Blow DM. 1969. Structure of crystalline a-chymotrypsin. 3. Crystallographic studies of substartes and inhibitors bound to the active site of a-chymotrypsin. J Mol Biol 46 337-348. 

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... 

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