Substrate selectivity, peptide

Lipid transfer peptides and proteins occur in eukaryotic and prokaryotic cells. In vitro they possess the ability to transfer phospholipids between lipid membranes. Plant lipid transfer peptides are unspecific in their substrate selectivity. They bind phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and glycolipids. Some of these peptides have shown antifungal activity in vitro The sequences of lipid transfer proteins and peptides contain 91-95 amino acids, are basic, and have eight cysteine residues forming four disulfide bonds. They do not contain tryptophan residues. About 40% of the sequence adopts a helical structure with helices linked via disulfide bonds. The tertiary structure comprises four a-helices. The three-dimensional structure of a lipid transfer peptide from H. vulgare in complex with palmitate has been solved by NMR. In this structure the fatty acid is caged in a hydrophobic cavity formed by the helices. 

Lowe et al. investigated the substrate selectivity of the proteases papain and chy-motrypsin using PEGA-bound combinatorial peptide hbraries (32) (Fig. 10.3) [24]. The quahtative extent of enzymatic cleavage of the resin-bound peptide in case of an accepted substrate strand was rapidly visualized by a significant reduction in the fluorescence of the beads visuahzed with a fluorescence microscope. Furthermore it was proven that substrate selectivity of the enzyme remain the same on the solid phase and in solution. 

Thus, for both the terminal diammonium and dicarboxylate substrates, selective binding by the appropriate receptors describes a linear recognition process based on length complementarity in a ditopic binding mode. Important biological species, such as polyamines, amino acid and peptide diamines, and dicarboxylates [4.18] may also be bound selectively. Recognition is achieved by multiple coordination to metal ions in dinuclear bis-macrocyclic coreceptors that complex selectively complementary bis-imidazole substrates of compatible length [4.21]. 

In addition to selecting peptides for metal substrates, phage display methods successfully identified peptides capable of forming sihea nanospheres. Recently, many groups have isolated proteins and peptides responsible for the in vivo prodnetion of sihea. Stone and coworkers have isolated additional novel peptides nsing phage display that are capable... 

Analogously, it will be important to evaluate cathepsin L-generated peptide products that serve as substrates for the subsequent aminopeptidase B (AP-B) step. Significantly, the AP-B substrates contain basic residues at their N-termini these substrates do not contain basic residues at their C-terminal as CPE substrates do. Therefore, although the anhydrotrypsin affinity column can be used to isolated CPE substrates, selective analyses of AP-B peptide substrates will require other approaches for isolation and analyses. It will be of interest in future studies to characterize AP-B neuropeptide substrates generated by secretory vesicle cathepsin L. 

Abdul-Hay SO, Lane AL, Caulfield TR, Claussin C, Bertrand J, Masson A et al (2013) Optimization of peptide hydroxamate inhibitors of insulin-degrading enzyme reveals marked substrate-selectivity. J Med Chem 56 2246-2255... 

The use of enzymes in non-aqueous solvents can be an advantageous alternative to reactions in water, especially for poorly water-soluble substrates and products, e. g. in the synthesis of esters, lactones or selected peptides. However, the knowledge of how solvents influence enzyme activity and selectivity is still not profound. As it is impossible to cover all relevant aspects within this chapter, the reader is referred to some selected articles for further reading115, 64 751. Here some of the basics will be discussed briefly in Sect. 7.5.4 reactor concepts for the use of enzymes in organic solvents will be presented. 

We have been involved in the synthesis of metal-based enzyme-like catalysts for hydrolysis of peptide bonds of proteins. We have been interested in hydrolysis as the target reaction since only one molecule is involved as the substrate, except for the water molecule. We selected peptide bonds as the targets since protein hydrolysis is important in the era of genomics. Proteomics and peptide bonds are quite stable with a half-life (26, 27) of spontaneous hydrolysis at (pH 7 at 25°C)... 

IV. PEPTIDE- OR PROTEIN-HYDROLYZING AGENTS LACKING SUBSTRATE SELECTIVITY... 

Since metal complexes with high activity for peptide hydrolysis were secured, it was subsequently attempted to achieve substrate selectivity in the hydrolysis of peptide bonds by the metal complexes. The active site of BB was constructed on the surface of partially chloromethylated cross-linked polystyrene (PCPS) (112). Here, the active site was chiral since L-arginine was used to introduce the guanidinium portion. 

Multinuclear metallocatalyst BJ manifested both catalytic activity and substrate selectivity in the hydrolysis of small peptides. The metal centers of the artificial active site of BJ were utilized both in substrate recognition and in catalytic conversion. The structure of the active site obtained by using the bowlshaped molecule, however, is unknown. In addition, it is not possible to synthesize a variety of artificial multinuclear metalloenzymes by the method of transferring catalytic elements confined in a prebuilt cage to a synthetic polymer. 

If a protein-cleaving catalyst based on a metal complex contains a recognition site selective for the protein substrate, the agent can manifest substrate selectivity. In the complex formed between the catalyst and the protein, the metal complex should take a highly productive position in order to possess a high effective molarity and, consequently, cleave the peptide bond effectively. 

---