Enzyme deactivation chymotrypsin

Fig. 25.2. Analysis of the catalytic activity and the inactivation of a-chymotrypsin at the single-molecule level, (a) Detection of single enzymatic turnover events of a-chymotrpysin. The fluorogenic substrate (suc-AAPF)2-rhodamine 110 is hydrolyzed by a-chymotrypsin, yielding the highly fluorescent dye rhodamine 110. (b) Representative intensity time trace for an individual a-chymotrypsin molecule undergoing spontaneous inactivation imder reaction conditions, (c) Inactivation trace for the intensity time transient in (b), obtained by counting the amount of turnover peaks in (b) in 10 s intervals. After approximately 1000 s, the enzyme deactivates through a transient phase with discrete active and inactive states, (d) Proposed model for the inactivation process. An initial active state is in equilibrium with an inactive state. This inactive state converts to another inactive state irreversibly whereby the corresponding active state has a lower activity than the previous one. All the transitions involved have energy barriers that can be overcome spontaneously at room temperature...

Exercise 25-30 The proteolytic enzyme, papain, differs from chymotrypsin in having cysteine, or a labile derivative thereof, as part of its active site. The enzyme is deactivated by substances that form complexes with, or react with, —SH groups and the activity is restored by reactions expected to regenerate an —SH group. Work out a schematic mechanism for cleavage of a peptide chain with papain that involves acylation of the critical —SH group of papain. 

The biocatalyst a-chymotrypsin s ability to hydrolyze 20 is inhibited in the presence of copolymer 19a loaded with 0.2 mol% of the triphenyl carbinol units. 47b Photoirradiation of 19a results in heterolytic bond cleavage and the formation of the cationic copolymer 19b. In this polymer structure, the biocatalyzed hydrolysis of 20 is activated (V = 1.0 pM min-1). The polymer-induced photostimulated activation and deactivation of a-chymotrypsin in the different membrane environments correlates with the permeability and transport properties of the substrate 20 through the different structures of the polymer membranes.1471 Flow dialysis experiments showed that the polymer states 17a, 18a, and 19a are nonpermeable to 20, and hence the biocata-lytic functions of the immobilized enzyme are blocked. The polymer structures 17b,... 

In spite of a long-time paradigm that enzymes can be active only in their natural aqueous media and other solvents cause deactivation and denaturation of proteins, at present a growing number of investigations are devoted to enzymatic reactions in organic solvents (Klibanov, 2001 Ke et al., 1996 Koskinen and Klibanov, 1996 and references therein). Such enzymes as a-chymotrypsin, subtilisin ribonuclease, pancreatuc lipase, and horse radish peroxidase have been found to be markedly active in organic solvents (alcohols, amines, tiols,anhydrous alkanes, acetonitril, dichloromethane, methyl acetate, etc.). 

Another approach to enzyme inhibition is to manipulate the pH to inactivate local digestive enzymes. Lee et al. conducted studies demonstrating that oral absorption properties of salmon calcitonin can be modulated by changing intestinal pH. Reducing the intestinal pH in the GI tract, increased absorption of the intact peptide.34 A sufficient amount of a pH-lowering buffer that lowers local intestinal pH to values below 4.5 can deactivate trypsin, chymotrypsin, and elastase. 

Furthermore, the stability of a-chymotrypsin in [EMIM][(CF3S02)2N] was studied by De Diego et al. Results were compared to those obtained in 1-propanol, a deactivating medium, and an aqueous solution of sorbitol, an enzyme-stabihzing medium. Using fluorescence and circular dichroism studies they showed that of the solvents used only the ionic liquid was able to stabilize the enzyme via the formation of a flexible and more compact 3D structure [41]. 

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