Chymotrypsin terminal groups

In a similar fashion, the Rotello group reported several protein-mediated assemblies of nanoparticles (Srivastava, Verma, et al. 2005 Verma et al. 2005). Unstable proteins such as chymotrypsin are readily denatured upon prolonged interaction with functionalized nanoparticles because of the exposure of proteins to the hydrophobic layer (Srivastava, Verma, et al. 2005). Addition of a hydrophilic portion to the monolayer by inserting a short tetraethylene glycol between the charged terminal group and hydro-phobic aliphatic chain circumvented this denaturation problem (Hong et al. 2004). 

Exercise 25-19 Eledoisin is a peptide isolated from the salivary glands of eledone, a Mediterranean eight-armed cephalopod. The peptide is a powerful hypotensive agent. Deduce a possible structure from the following information (1) Complete hydrolysis gives equal amounts of ammonia, Ala, Asp, Glu, Gly, lie, Leu, Lys, Met, Phe, Pro, and Ser. (2) No free amino /V-terminal group or free carboxyl C-terminal group can be detected. (3) Chymotrypsin hydrolysis forms two peptides, L and M. Their compositions are... 

The newly formed amino-terminal group of isoleucine 16 turns inward and forms an ionic bond with aspartate 194 in the interior of the chymotrypsin molecule (Figure 10.33). Protonation of this amino group stabilizes the active form of chymotrypsin. 

Kurosky, A., and Hofmann, T., 1972, Kinetics of the reaction of nitrous acid with model compounds and proteins, and the conformational state of N-terminal groups in the chymotrypsin family, Canad. J. Biochem. 50 1282. 

The general answer appears to be that chymotrypsin and, indeed, many other peptidases, are synthesized in the pancreas as inactive proenzymes (or zymogens), which are incapable of carrying out their catalytic function, until they are secreted into the intestine. Once in the intestine, the blocking (usually terminal) groups are hydrolyzed off and the active enzyme is produced. 

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide .

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.

In another successful case, Hexter and Westheimer (1971) were able to locate 5% of the total radioactivity in Tyr-146 after irradiation of [14C]diazoacetylchymotrypsin. The reaction is actually intermolecular, occurring in chymotrypsin dimers. Westheimer s group have determined the structure of several of the modified amino acids derived from the photolysis of proteolytic enzymes acylated with diazo reagents. Such data is not available for other photoaffinity reagents. Knowing that O-carboxy-methyl tyrosine was an expected insertion product Hexter and Westheimer (1971) were able to show that of the two Tyr residues in the chymotrypsin B chain only Tyr-146, the C-terminal residue, was modified. If the nature of the modified amino acid had not been known it would have been considerably more difficult to pin-point the site of photolabeling. 

Peptides that display a terminally located aldehyde function in their structure constitute another group of modified peptide enzyme inhibitors. The sequence benzyloxycarbonyl-Pro-Phe-CHO fulfils the known primary and secondary specificity requirements of chymotrypsin and has been found to be a potent reversible inhibitor of this proteolytic enzyme (Walker et al. 1993). Further, protease inhibitors comprising terminally located aldehyde function are antipain, leupeptin, chymostatin, and elastatinal. In addition, also phos-phoramidon, bestatin, puromycin, and amastatin represent modified peptides which can reversibly inhibit enzymes. 

Chymotrypsin does not hydrolyze extensively bonds formed by the imino group of proline. Bonds of this type were not cleaved in ribo-nuclease, c3rtochrome c, and the a-chain of human hemoglobin. The -Phe-Pro- bond in ovine corticotropin has been reported to be hydrolyzed by chymotrypsin. The extent of hydrolysis was low as judged by the 6 % yield of the peptide which contained the carboxyl-terminal phenylalanine of the -Phe-Pro- bond. 

For the N-terminal deprotection of peptides, the enzyme penicillin G acylase from E. coli has been applied. This attacks phenylacetic acid (PhAc) amides and esters but does not hydrolyze peptide bonds [12-14,25]. The danger of a competitive cleavage of the peptide backbone at an undesired site, which always exists when proteases like trypsin and chymotrypsin are used, is overcome by using the acylase. The penicillin G acylase accepts a broad range of protected dipeptides (27) as substrates, and selectively liberates the N-terminal amino group under almost neutral conditions (pH 7-8, room temperature), leaving the peptide bonds as well as the C-terminal methyl-, allyl-, benzyl-, and tert-butyl ester unaffected (Fig. 8) [25a,bj. On the other hand, the phenylacetamide... 

Enzymes from several different endopeptidase classes have been shown to perform this bond cleavage. Initially a chymotrypsin, serine protease, was proposed [1] and two groups have reported work on the design of inhibitors of the action of this type of enzyme (see later). It has been demonstrated, however, that a chymotrypsin-type enzyme does not cleave big ET-1 cleanly to only an active ET-1 (1-21) fragment and a stable C-terminal fragment, as cleavage can occur both initially at Tyr -Gly and subsequently to break down the ET-1 formed [61]. 

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