Substrates peptides

Kinetic studies with pepsin have produced bell-shaped curves for a variety of substrate peptides see below, (a). 

Several classes of non-covalent substrate based inhibitors have been reported, and are grouped below based on the nature of the C-terminal group interacting with the catalytic triad of the enzyme. The majority of the reported inhibitors are based on the N-terminal product of a modified substrate of the NS5A/5B cleavage side or to a lesser extent of the NS4A/4B substrate peptide. 

After the discovery of weak inhibition by the N-terminal products of substrate peptides, researchers at Boehringer Ingelheim reported potent and specific hexapeptides providing up to a 10 -fold increase in potency (Figure 2.3) [89]. 

Since kinases are not hydrolytic enzymes, a small molecule-based FRET probe does not seem to be a straight forward solution for this enzyme activity. Nevertheless, quite a number of fluorescent probes based on small substrate peptides have been prepared in... 

I/cBa substrate peptide (Figure 7.11). These isolated j5-catenin and I/cBa peptides should accurately reflect the context of these destruction motifs in their respective full-length proteins, since Lysl9 and the destruction motif of j5-catenin are both in a 133-residue N-terminal region that has been previously shown to have a disordered structure by proteolytic digestion analysis [104]. The destruction motif of I/cBa similarly resides outside the structured ankyrin-repeat domain. 

The crystal structure of the peptide substrate-binding domain (140—245 of 517 residues of human al subunit) of the human type I enzyme forms 2.5 tetratricopeptide (TPR) repeat domains with five a helices (PDB accession number ITJC). The organization of tyrosine residues is suggested to be key to its interaction with the substrate peptide in a polyproline II helix. The TPR motif is composed of a 34 amino acid repeated a helical motif, and is typically involved in protein-protein interactions. The tandem repeats of TPR motifs are found in many proteins related to chaperone, cell cycle, transcription, and protein transport... 

Photocrosslinkers have been incorporated in E. coli to confirm close contact between specific residues of a protein and its substrate. ClpB, a heat shock protein that aids in the disaggregation and refolding of proteins during the heat shock response, has a conserved aromatic residue (Tyr251) in the central pore of its AAA+ domain, considered to be the main substrate recognition residue. " After Tyr251 in ClpB was replaced with />BpA, biotinylated substrate peptides were shown to be crosslinked upon UV light exposure, but not if BpA was incorporated elsewhere in the AAA+ domain of this protein. 

Tethering with extenders was also used to identify inhibitors to the antiinflammatory target caspase-1 [28, 29]. In this case, one of the same extenders previously designed for caspase-3 selected an entirely different set of fragments. This is consistent with different substrate peptide sequence preferences WEHD for caspase-1 vs DEVD for caspase-3 [30]. 

The active center of trypsin is shown in Fig. 2. A serine residue in the enzyme (Ser-195), supported by a histidine residue and an aspartate residue (His-57, Asp-102), nucle-ophilically attacks the bond that is to be cleaved (red arrow). The cleavage site in the substrate peptide is located on the C-terminal side of a lysine residue, the side chain of which is fixed in a special binding pocket of the enzyme (left) during catalysis (see p. 94). 

Zhang, X. and Cheng, X. (2003) Structure of the Predominant Protein Arginine Methyltransferase PRMTl and Analysis of Its Binding to Substrate Peptides. Structure (Camb), 11 (5), 509-520. 

Figure 3-2. Reaction mechanism of chymotrypsin as an example of covalent catalysis. Step I involves attack of the enzyme s active site serine on the peptide bond to be cleaved. In step II, a covalent complex is formed between the enzyme and a portion of the substrate (peptide 2) with release of the rest of the substrate (peptide I). Step III involves hydrolysis of the enzyme-substrate complex, which releases peptide 2 and completes the reaction.

ELISA has been used for measuring caspase activity. For the ELISA of intracellular caspase activity at the very early stages of apoptosis, apoptotic cells are first lysed to isolate their intracellular contents. Different caspase activities in the cell lysate can then be determined by the addition of a caspase-specific tetrapeptide substrate that is conjugated to the color reporter molecule p-nitroanilide (pNA) (e.g., DEVD-pNA for caspase-3 and lETD-pNA for caspase-8). The cleavage of the substrate peptide by the caspase releases the chromophore pNA, which can be... 

Hu BH, Messersmith PB (2003) Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels. J Am Chem Soc 125 14298-14299... 

If an asymmetric hydrogenation of C=C bonds is desired in the presence of achiral catalysts, chiral information is required to be present in the substrate. Peptides and cyclopeptides containing dehydroaminos acid units are very good substrates achieving quite high stereoselectivities upon asymmetric hydogenation on 10% Pd-C or other achiral catalysts 49 841. 

Fig. 8.11. Recognition of phosphotyrosine-containing substrate peptides by SH2 domains of Src kinase and phospholipase Cyl. Binding of phosphotyrosine-containing peptides to SH2 is shown schematically, based on crystal structures of the complexes. The SH2 domain of Src kinase has a basic binding pocket for the phosphotyrosine residue and a hydrophobic pocket for the isoleucine residues at position -1-3 of the peptide substrate. The SH2 domain of PL-Cyl has a hydrophobic binding surface to which the C-terminal part of the peptide P-Tyr-Ile-Ile-Pro-Leu-Pro-Asp binds. According to Cohen, (1995).

A catalytic mechanism (Figure 14) was proposed on the basis of kinetic, spectroscopic and crystallographic data . The substrate peptide binds to Zn2 with its N-terminal amino group and to Znj with the carbonyl oxygen of the scissile peptide bond. Additionally, the N-terminus interacts with Aspl79. Upon substrate binding, the bridging water... 

Fig. 14.1. Ribbon structure (magenta) of the phosphorylase kinase crystal structure 2PHK (20) bound with ATP (green carbons, colored by atom type) and substrate peptide (light blue ribbon). The N- and C-terminal lobes are highlighted the hinge region is shown in cyan, the a-C helix in gray, and the -loop in orange.

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