Histidine peptides, hydrolysis

Effect of Various Histidine Peptides on the Rate of Hydrolysis of p-Nitrophenyl Acetate at pH 7.73 ... 

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.

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. ... 

Amino-acid Protection. - Carboxyamidomethyl (CAM) esters have been shown to be useful protecting groups in a-chymotrypsin and papain-catalysed peptide hydrolysis and synthesis.5 10 1,3-Dioxans (582) can be obtained from Na-protected serine derivatives by acid-catalysed transacetalation and are sufficiently robust to survive both amino deprotection and peptide coupling reactions. 11 (L)-Histidine benzyl ester can be prepared as the ditosylate salt... 

As an example of application to a reaction involving a macromolecule, we indicate below the transition state obtained by the combined classical quantum force field in a study of the peptide hydrolysis reaction catalyzed by thermolysin. The subsystem is limited to the substrate, a water molecule, a zinc atom, the side chains of His 143, Glul66, the whole glutamic acid and the histidine 146 moieties (see Figure 10). The transition state represented here corresponds to the proton transfer from Glul43 to the nitrogen atom of the peptidic bond before the breaking of the bond. 

Selective cleavage of peptides and proteins is an important procedure in biochemistry and molecular biology. The half-life for the uncatalyzed hydrolysis of amide bonds is 350 500 years at room temperature and pH 4 8. Clearly, efficient methods of cleavage are needed. Despite their great catalytic power and selectivity to sequence, proteinases have some disadvantages. Peptides 420,423,424,426 an(j proteins428 429 can be hydrolytically cleaved near histidine and methionine residues with several palladium(II) aqua complexes, often with catalytic turnover. 

In related work a library of 1,458 peptide ligands and various metal salts was tested in hydrolysis reactions of (p-nitrophenyl)phosphates.35 An active substructure composed of polymer-bound histidine in combination with Eu3+ was identified by further dissecting the original hit structure. It needs to be pointed out that catalytically active polymer beads can also be tested for catalytic activity using IR-thermography. In a seminal paper this was demonstrated using 7,000 encoded polymer beads prepared by split-and-pool methods, specifically in the metal-free acylation of alcohols.36... 

Fig. 3.6. Stereoelectronic control of the cleavage of the tetrahedral intermediate during hydrolysis of a peptide bond by a serine hydrolase. The thin lines represent the reactive groups of the enzyme (serine, imidazole ring of histidine) the thick lines represent the tetrahedral intermediate of the transition state. The full circles are O-atoms open circles are N-atoms. The dotted lines represent H-bonds the thick double arrow indicates an unfavorable dipole-dipole interaction [21]. A (R)-configured N-center B (S)-configured N-center.

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

Flg.1. In the amino acid sequence of KO-42 is encoded its fold and its function as it controls the formation of a hairpin helix-loop-helix motif that dimerizes to form a four-helix bundle. On the surface of the folded motif a reactive site is formed that catalyzes hydrolysis, transesterification and amidation reactions of reactive esters, whereas unfolded peptides are incapable of cooperative catalysis. In addition the values, and thus the reactivities, of the histidine residues are controlled by the fold. The pK of each His residue of KO-42 is shown in the figure and deviate by as much as 1.2 units from that of random coil peptides which is 6.4... 

The 42-residue peptide KO-42 folds in solution into a hairpin helix-loop-helix motif that dimerizes to form a four-helix bundle. On the surface of the folded motif there are six histidines with assigned piC values in the range 5.2 to 7.2 (Fig. 1) and the second-order rate constant for the hydrolysis of mono-p-nitro-phenyl fumarate is 1140 times larger than that of the 4-methylimidazole-cataly-zed reaction at pH 4.1 and 290 K [13]. The reaction mechanism was found to be pH dependent as the kinetic solvent isotope effect was 2.0 at pH 4.7 and 1.0 at pH 6.1 and the pH dependence showed that the reaction rate depended on residues in their unprotonated form with piCj, values around 5. It was thus established that there are functional cooperative reactive sites that contain protonated and unprotonated His residues. 

The mechanism of catalysis by these enzymes has been extensively investigated (for review see ref. 10). Essentially, the active site serine via its side chain hydroxyl group performs a nucleophilic attack on the carbonyl carbon of the scissile peptide bond thus forming a tetrahedral intermediate. A histidine residue in the active site serves as a general base accepting the proton from the serine residue. The acyl enzyme thus formed is broken down via a nucleophilic attack of a water molecule to complete the hydrolysis of the peptide bond. 

The cleavage mechanism of the caspases is shown schematically in Fig. 15.5. They use a typical protease mechanism with a catalytic diad for cleavage of the peptide bond. The nucleophilic thiol of an essential Cys residue forms a covalent thioacyl bond to the substrate during the catalysis. The imidazole ring of an essential histidine is also involved in catalysis and this facilitates hydrolysis of the amide bond in the sense of an acid/base catalysis. 

Only very few among the common amino acids possess a pK within the range 5.8-7.0. Therefore, the imidazole ring of histidine was suspected very early to represent the group responsible for nucleophilic attack on the substrate (38). The pK of free imidazol is 6.9 (39) that of imidazol, contained in histidine or its peptides, varies between 5.6 and 7.1 (40). Imidazol is well known to form unstable acyl derivatives, which undergo spontaneous hydrolysis because of the presence of the resonating triad unit —-N—C= N— (41). In addition, imidazol and its derivatives catalyze the hydrolysis of certain esters, especially those derived from phenols (42). Likewise, the behavior of imidazol towards thio esters reflects exactly the specificity of ChE s (see IV, 4). Thus, thiol esters are split (43), whereas thiono esters are resistant (21). 

Activation reactions catalyzed by serine proteases (including kallikreins) are an example of limited proteolysis in which the hydrolysis is limited to one or two particular peptide bonds. Hydrolysis of peptide bonds starts with the oxygen atom of the hydroxyl group of the serine residue that attacks the carbonyl carbon atom of the susceptible peptide bond. At the same time, the serine transfers a proton first to the histidine residue of the catalytic triad and then to the nitrogen atom of the susceptible peptide bond, which is then cleaved and released. The other part of the substrate is now covalently bound to the serine by an ester bond. The charge that develops at this stage is partially neutralized by the third (asparate) residue of the catalytic triad. This process is followed by deacylation, in which the histidine draws a... 

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