Histidine residues chymotrypsin

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

In chymotrypsin and subtilisin, this charge-relay network system, as it is called, is made up of a specific aspartic acid residue, acting as B e, and a specific histidine residue (acting as the amphoteric B—A—H) ... 

The facile reaction of CAA and BAA with nucleosides and nucleotides is one example of many of the applications of the bifunctional reactivity of halogenated aldehydes and ketones in modification of biomolecules. In an early example of the extensive use of halogenated ketones as protease substrate analogues, l-V-tosylamido-2-phenylethyl chloro-methyl ketone (TPCK) 30 was synthesized as a chymotrypsin substrate analogue. Stoichiometric inhibition was accompanied by loss of one histidine residue as a result of alkylation by the chloromethyl moiety68. A host of similar analogues were subsequently prepared and used as selective enzyme inhibitors, in particular for the identification of amino acid residues located at enzyme active sites69. 

Imidazole is found in many enzymes as histidine residue. It plays a role of electron donor in biological systems. Imidazole is one of the nucleophiles, and many nucleophile-containing polymers have been synthesized as a mode of the enzyme such as a-chymotrypsin. 

This sort of problem underlines the need for an unequivocal determination of microscopic ionization constants. As already mentioned, the rate-limiting step in catalysis is controlled by an ionization with a pKa of 7. Cruickshank and Kaplan (30) used a-chymotrypsin as a test enzyme and estimated the pKa of its two histidine residues from the pH dependence of their reaction with trace amounts of tritiated l-fluoro-2,4-dinitro-benzene. From their data, a pKa of 6.8 was assigned to His-57 and 6.7 to His-40, which is not involved in catalysis. This data is consistent with deprotonation of His-57 being the critical ionization for catalysis. 

Affinity labels are molecules that are structurally similar to the substrate for the enzyme that covalently modify active site residues. They are thus more specific for the enzyme active site than are group-specific reagents. Tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analog for chymotrypsin (Figure 8.21). TPCK binds at the active site and then reacts irreversibly with a histidine residue at that site, inhibiting the enzyme. The compound 3-bromoacetol is an affinity label for the enzyme triose phosphate isomerase (TIM). It mimics the normal substrate, dihydroxyacetone phosphate, by binding at the active site then it covalently modifies the enzyme such that the enzyme is irreversibly inhibited (Figure 8.22). 

Figure 8.21. Affinity Labeling. (A) Tosy 1-1-phenylalanine chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. (B) TPCK binds at the active site of chymotrypsin and modifies an essential histidine residue.

General acid-base catalysis. In general acid-base catalysis, a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine (Section 9.1.3). 

The strategy used by the cysteine proteases is most similar to that used by the chymotrypsin family. In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the nucleophile that attacks the peptide bond (see Figure 9.18). in a manner quite analogous to that of the serine residue in serine proteases. An ideal example of these proteins is papain, an enzyme purified from the fruit of the papaya. Mammalian proteases homologous to papain have been discovered, most notably the cathepsins, proteins having a role in the immune and other systems. The cysteine-based active site arose independently at least twice in the course of evolution the caspases, enzymes that play a major role in apoptosis (Section 2.4.3). have active sites similar to that of papain, but their overall structures are unrelated. 

Figure 9.8. Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis. The dashed green lines indicate favorable interactions between the negatively charged aspartate residue and the positively charged histidine residue, which make the histidine residue a more powerful base.

CE separation buffer. IMAGE was used to study surface-related affinity characteristics (number and accessibility of histidine residues and histidine microenvironment) for particular immobilized metal-ion chelate ligands in such proteins as cytochrome-c, ribonucle-ases A and B, chymotrypsin, and kallikrein [8],... 

Normally, an alcoholic group such as that on serine is not a good nucleophile. However, there is usually a histidine residue close by to catalyse the reaction. For example, the mechanism by which chymotrypsin hydrolyses peptide bonds is shown in Fig. 4.20. 

B) TPCK binds at the active site of chymotrypsin and modifies an essentia histidine residue. 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

In the past ten years, there has been developed a series of enzyme inhibitors that combine the features of an alkylating agent with specificity for the active site of an enzyme, thus permitting alkylation and identification of a group at or near the active center of an enzyme, or a particular enzyme to be specifically inactivated. Thus a l-chloro-4-phenyl-3-p-toluenesulfonamido-2-butanone ( W-p-tolylsulfonylphenylalanine chloro-methyl ketone ) inactivates chymotrypsin (which cleaves a peptide bond adjacent to an aromatic residue), and 7-amino-l-chloro-3-p-toluene-sulfonamido-2-heptanone ( a-iV-p-tolylsulfonyllysine chloromethyl ketone ) inhibits trypsin (which cleaves a peptide bond adjacent to lysine. In both cases, a histidine residue at the active site is alkylated, and neither inhibitor will inhibit the other enzyme at low concentrations. 

As with peptide hydrolysis, several enzyme systems exist that catalyze carboxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nucleophile in turn, serine may be activated by hydrogen-bond formation—or even proton abstraction—by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphory 1-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. In glucose-6-phosphatase the nucleophile has been proposed to be a histidine residue. ... 

Jornvall and Harris (91) presented data for the structures around all of the 14 cysteine residues in each protein chain. Analysis by Jornvall (92,93) of different peptide mixtures obtained after treatment of the protein with trypsin (before or after maleylation), chymotrypsin, pepsin, cyanogen bromide, or thermolysin yielded amino acid sequence information for all parts of the subunit and the primary structure of the whole protein chain was deduced (5S). It was found to contain 374 residues and is shown in Table I. An acetylated serine residue is at the N-terminus and the reactive cysteine residue is at position 46. Some residues are unevenly distributed (PS). Six of the seven histidine residues are in the N-terminal half of the molecule, the two tryptophan residues are in either terminal region, the four tyrosine residues are in the middle of the primary structure, and none of the 14 cysteine residues occur in the C-terminal quarter of the molecule. A characteristic distribution of hydrophobic residues was also noticed (93), which may now be partly correlated with the presence of large hydrophobic cores in the tertiary structure of the protein (Section II,C,3). Most regions of the primary structure were analyzed in many different overlapping peptides (92-9 ) with a corresponding increase in reliability. The structure is in excellent agreement with the total composition determined by acid hydrolysis (93). It is compatible with independently determined partial structures of... 

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