Scheme, proteolytic

Proteolytic scheme. The arrows indicate the specificities of the various peptidases. 

Figure 51-7. Scheme of sites of action of streptokinase, tissue plasminogen activator (t-PA), urokinase, plasminogen activator inhibitor, and Kj-antiplasmin (the last two proteins exert inhibitory actions). Streptokinase forms a complex with plasminogen, which exhibits proteolytic activity this cleaves some plasminogen to plasmin, initiating fibrinolysis. 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

In natural bioactive peptides the modes of cyclization described previously may be prevented either by the lack of suitable side-chain functionalities for lactamization or because these as well as the amino and carboxy termini are crucially involved in the bioactivity itself, and thus cannot be modified. In order to overcome these potential limitations, the concept of backbone cyclization has been proposed.129 According to this, the cyclization is performed by a covalent interconnection of two backbone amides by artificial spacers or of one backbone amide by a correctly functionalized spacer with side-chain functions or with the N- and C-terminus of the peptide (Scheme 21). This type of strategy significantly increases the diversity of possible ring structures (see Scheme 22) and of their related libraries (see Section 6.8.4). Its potential for enhancing the stability of the related peptide derivatives toward proteolytic digestion,[417 419 potency,141942" and selectivity,11417-419 is well-established. 

On the basis of this anti-proteolytic effect of sialic acids, a hypothetical model435 for the role of sialidase in clostridial infections is shown in Scheme 4. It is considered that the bacterial enzyme releases sialic acids from cell-surface glycoproteins of the infected tissue, which thereafter can be readily attacked by proteases. This cooperation between sialidase and protease may support the spreading of the bacteria. Acylneuraminate pyruvate-lyase, also shown in this model, degrades sialic acids for energy supply, and growth, of the bacteria. 

Fig. 6.4 Proposed scheme for ATj-mediated transactivation of the EGF receptor and the subsequent activation of ERK1/2. The interaction of angiotensin II with the ATi receptor stimulates several pathways that result in the transactivation of the EGF receptor. In one pathway, phospholipase C (PLC) is activated, which in turn promotes the production of reactive oxygen species (ROS). ATj receptor activation also elevates [Ca2+]j, contributing to the stimulation of a Ca2+-dependent tyrosine kinase, of which Src and proline-rich tyrosine kinase-2 (Pyk2) are the preferred targets. Both ROS and the Ca2+-dependent tyrosine kinase activate a metalloprotease, presumably ADAM17, which proteolytically activates the EGF ligand. The Ca2+-dependent tyrosine kinase, as well as a Ca2+-independent kinase, also phosphorylate the EGF receptor. The activation of the EGF receptor triggers a cascade leading to the activation of ERK1/2. EGF receptor transactivation also proceeds via a G-protein-independent pathway (see text).

For epitope elucidation of the polyclonal anti-c APP antibody, the analytical scheme summarised in Figure 3 was employed (Macht et al. 1996 Tian et al. 2005). The antibody was first immobilised on Sepharose embedded in a microcolumn (s. Materials and Methods). The synthetic peptide cAPP(724-770) was used as antigen and initially cleaved by trypsin and Glu-C protease, yielding the proteolytic fragments summarised in Table 1. 

Peptoids are oligomers of N-substituted glycines. Hence, the principal difference between peptides and peptoids is the location of the amino acid side chains with respect to the backbone. While in peptides 6 the side chains are attached to the a-carbon of the amino acids, in peptoids 7 they are linked to the amide nitrogens of the peptide backbone (Scheme 4) [42,43] Similar to amide-alkylated or reduced peptides (see Section 4.3.7.2.2), peptoids lack the peptide bond CO-NH, and are, therefore, less susceptible to proteolytic degradation. Consequently, peptoids are attractive molecules for the generation of combinatorial libraries. 

Fig. 4.6. The proposed arrangement of the RC and h/c, complex in the photosynthetic chain of Rps. sphaeroides. The scheme indicates the reduction of Q to QHj by a pair of RC complexes and the net oxidation of QH2 by two turnovers of the oxidoreductase, as a balance of the oxidation of two quinols and the reduction of one quinone at the site. The proposed sites of proteolytic reactions are also indicated (from Ref. 93).

All amino acid residues except Gly are chiral at the carhon hearing the side-chain and are subject to base-catalysed racemisation. Scheme 11.4 shows the mechanism involved. In alkaline solution the hydrogen of the a-methine is removed by base to form a carban-ion intermediate which can then generate D-enantiomers, which are nonmetabolisable, or create peptide bonds which are not broken down by proteolytic enz)mies. 

Fig. 5. The principle of processing-independent immunoassays. The scheme illustrates the processing from translation of pre-pro-protein in the endoplasmatic reticulum, via intermediates in the Golgi apparatus and early secretory vesicles, to final maturation (see also Fig. 3). Divisions within the protein bars indicate proteolytic cleavage sites. The protuberances on the tops and ends of the bars illustrate different forms of amino acid derivatization. The filled-in areas of the protein bars are the selected processing-independent sequences, toward which the processing-independent immunoassay is developed.

Tin hydride reagents are versatile tools for the functionalization of alkenes and alkynes. Based on this concept, Nicolaou and coworkers developed a polymer-bound tin hydride (118) that reacts via Pd-catalyzed hydrostannylation (or nucleophilic attack on the tin chloride with a vinyl lithium) with alkynes to give alkenylstannanes [116]. After further transformation to derivatives 119, the resin-bound substrates undergo proteolytic traceless cleavage to yield unsubstituted alkenes 120. Alternatively, the stannane can be employed for intramolecular Shlle coupling to produce macrolactones 121 in the cleavage step (Scheme 16.28). 

In addition to the above mentioned databases that try to cover the entire world of enzymes, there are a number of more topical databases focusing on particular enzyme families. The MEROPS database, maintained at the Babraham Institute in Cambridge, provides a catalog and a structure-based classification scheme for all proteolytic enzymes 58. In addition to the classification, the database also provides a digest of published information on the peptidases as well as dadograms and multiple sequence alignments of the peptidase families. 

The catalyst for cleavage of peptide deformylase was searched with a library of catalyst candidates synthesized by the Ugi reaction (Scheme 2) (134). In this multicomponent condensation reaction, the mixture of a carboxylic acid, an amine, an aldehyde, and an isocyanide produces an iV-acyl amino acid amide. The catalyst candidates, therefore, are iV-acylamino acid amides containing various polar and nonpolar pendants as well as the Co(III) complex of cyclen. The Co(III) complex of cyclen (135) was chosen as the proteolytic center in view of the results described in Section V.A. Cyclen with three secondary amines protected with ferf-butyloxycarbonyl (t-boc) groups was incorporated in either the carboxyl or the amine component of the Ugi reaction. Later, the t-boc groups were removed and Co(III) ion was inserted to the cyclen portion. 

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