Chymotrypsin stability

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

De Diego T, Lozano P, Gmouh S et al (2004) Fluorescence and CD spectroscopic analysis of the a-chymotrypsin stabilization by the ionic liquid, l-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide. Biotechnol Bioeng 88 916-924... 

Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. 

For many serine and cysteine peptidases catalysis first involves formation of a complex known as an acyl intermediate. An essential residue is required to stabilize this intermediate by helping to form the oxyanion hole. In cathepsin B a glutamine performs this role and sometimes a catalytic tetrad (Gin, Cys, His, Asn) is referred too. In chymotrypsin, a glycine is essential for stabilizing the oxyanion hole. 

To realize the reason for this result from a simple intuitive point of view it is important to recognize that the ionized form of Aspc is more stable in the protein-active site than in water, due to its stabilization by three hydrogen bonds (Fig. 7.7). This point is clear from the fact that the observed pKa of the acid is around 3 in chymotrypsin, while it is around 4 in solution. As the stability of the negative charge on Aspc increases, the propensity for a proton transfer from Hisc to Aspc decreases. 

It is generally accepted that Na ions can be occluded in E]P forms. Occlusion of SNa ions per EP has been demonstrated in chymotrypsin cleaved enzyme and in the Cr-ADP-EiP[3Na] complex [29]. Three Na ions can also be occluded per EP in a complex stabilized by oligomycin in the absence of Mg or phosphate [97] while a maximum of two Na ions are occluded per a subunit in the ouabain complex. 

Powders for Reconstitution. Drugs that have only limited stability in liquid form are prepared as sterile powders for reconstitution by the pharmacist prior to dispensing to the patient. In ophthalmology, these drugs include a-chymotrypsin, echothiophate iodide... 

E.V. Kudryashova, A.K. Gladilin, A.V. Vakurov, F. Heitz, A.V. Levashov, and V.V. Mozhaev, Enzyme-poly electrolyte complexes in water-ethanol mixtures negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects. Biotechnol. Bioeng. 55, 267-277 (1997). 

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. 

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... 

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.

Resistance to peptidases was also reported for the octapeptoid 6.106 when incubated with papain, chymotrypsin, or thermolysin [229], However, resistance to peptidases may not be synonymous with a long half-life in vivo, since many factors beside peptidases can be expected to contribute to the elimination of peptoids. An indirect indication of this effect can be found for antimicrobial peptoids and particularly compound CHIR29498 (6.107) [232], In mice infected with Staphyllococus aureus, this peptoid was less active when injected 2 h post-infection compared to 0 h or 0.5 h. The conclusion drawn by the authors was that the compound requires optimization for improved absorption or stability within the body. 

Fig. 54. An asparagine side chain making a hydrogen-bond to the main chain NH of residue n + 2, an arrangement which helps stabilize the central peptide of a tight turn. Residues 91-93 from chymotrypsin.

Despite their lack of stabilizing disulfide bridges Potl inhibitors feature a common, stable fold. The N-terminus is coiled, although in some structures a small /3-strand has been identified. After a turn the structure adopts an a-helical structure, followed by a turn and an other /3-strand. The sequence then features an extended turn or loop motif that contains the reactive site of the inhibitor before it proceeds with a /3-strand running almost parallel to the /3-strand after the a-helix. After another turn and coiled motif a short /3-strand antiparallel to the other /3-strands precedes the coiled C-terminus. Usually the N-terminal residue in the reactive site is an acidic residue followed by an aromatic amino acid, that is, tyrosine or phenylalanine. Figure 11 shows the complex of chymotrypsin inhibitor (Cl) 2 with subtilisin, the hexamer of Cl 2 from H. vulgare and a structural comparison with a trypsin inhibitor from Linum usitatissimum ... 

Numerous suggestions have been made that enzymes might owe part of their catalytic efficiency to the opportunity they afford for stabilization of intermediates or transition states by hydrogen bonding to functional groups near the active site. For example, in the case of (x-chymotrypsin this might be represented as in [43] where... 

In a recent study of protease oc-chymotrypsin transesterification reactions, several ionic liquids ([EMIM]BF4, [EMIM]Tf2N, [BMIM]BF4, [BMIM]PF6, and [MTOA]Tf2N) were used as the isolation media for the enzyme. Among these ionic liquids, [EMIM]BF4 showed the least activity, and [BMIMJPF offered the best stabilization of the enzyme (281). 

Fig. 2. Model image of a typical substrate bound to ot-Chymotrypsin. (a) Binding of the substrate, (b) Three additional hydrogen bonds stabilize the intermediate oxyanion.

Multipoint attachment to a support protects the enzyme from inactivation by organic solvents. Mozhaev et al. (1990) have recently demonstrated that covalent linkage to polyacrylamide gel stabilizes df-chymotrypsin from denatmation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. 

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... 

Fig. Z14. The activation of chymotrypsin via proteolytic cleavage, a) Chymotrypsinogen is transformed into the active forms of chymotrypsin n and a by trypsin and autoproteolysis, b) The N-terminal isoleucine residue Ile6 is particularly important for the activity of chymotrypsin. The positively charge NH2 group of llel6 interacts electrostatically with Aspl94 and stabilizes an active conformation of the catalytic center. After Stryer Biochemistry , with permission.

Proteases have received less attention than lipases, but in one of the earliest papers on biocatalysis in ionic liquids it was noted that the activity loss of thermo-lysin during preincubation proceeded much more slowly in [BMIm][PF6] than in ethyl acetate [8]. The storage stability of a-chymotrypsin in the ionic liquid [EMIm][ Tf2N] was compared with that in water, 3 M sorbitol, and 1-propanol. The residual hydrolytic activity (after dilution with aqueous buffer) was measured vs time, and structural changes were monitored by fluorescence and CD spectroscopy as well as DSC [98]. The enzyme s life-time in [EMIm][ Tf2N] at 30°C was more than twice that in 3 M sorbitol, six times as long as that in water, and 96 times as long as that in 1-propanol. 

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional structure of chymotrypsin is shown in Figure 6-18, with functional groups in the active site emphasized. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. 

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