Peptidases substrate specificity

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

The substrate specificity of the type I signal peptidases is known as the ( — 3, —1) rule observed at the c-region of signal peptides (von Heijne, 1984 Jain et al., 1994), where the residues at positions —3 and -1 from the cleavage site (i.e., cleavage occurs at the peptide bond between —1/ + 1 positions) are usually small (and neutral) residues, such as alanine. Recently, the x-ray crystallographic structure of the signal... 

The above examples illustrate the versatility and overlapping substrate specificities of peptidases, but they also serve to explain the difficulties faced by medicinal chemists who try to design bioactive peptides that have improved pharmacokinetic properties. Clearly, general predictive rules and a global understanding of the in vivo fate of peptides are not in sight, but the sections below will show that medicinal chemists have developed various successful strategies of a rather empirical nature [7][181-188],... 

This endopeptidase [EC 3.4.21.20], a member of the peptidase family SI, has substrate specificity similar to that of chymotrypsin C. 

This peptidase family Cl enzyme [EC 3.4.22.27] is a lysosomal cysteinyl-dependent endopeptidase with substrate specificity similar to cathepsin L. 

Major hydrolysis reactions are ester and amide hydrolysis. These are catalyzed by a group of enzymes with overlapping substrate specificity and activity. Hydrazides can also undergo hydrolysis. Some of the newer drugs such as hormones, growth factors, and cytokines now being produced are peptides, and certain toxins are also peptides or proteins, so the role of peptidases may be important. 

A recent report describes the presence of pyrrolidone carboxylyl peptidase in rat liver (46). A partial purification of the enzyme has been obtained by ammonium sulfate fractionation and gel filtration on Sepha-dex G-200. The rat liver enzyme is similar to that obtained from Pseudomonas thus, it is protected against inactivation by 2-pyrrolidone and exhibits similar substrate specificity. 

Horsthemke B, Bauer K (1982) Substrate specificity of an adenohypophyseal endopeptidase capable of hydrolyzing LHRH preferential cleavage of peptide bonds involving the carboxyl terminus of lower of it of hydrophobic and basic amino acids. Biochemistry 21 1033-1036 Horsthemke B, Knisataschek H, Rivier J, Sandow J, Bauer K (1981) Degradation of LHRH and analogs by adenohypophyseal peptidases. Biochem biophys Res Commun 100 753-759... 

Many enzymes are not exactly substrate-specific, e.g. peptidases acting as hydrolases, esterases etc. to some extent (Vallee and Williams 1968) or vice versa. 

Last but not least, it should be mentioned that a couple of peptidases have industrial importance. In particular, since subtilisins have a broad substrate specificity and are highly stable at neutral and alkaline pH they are of considerable industrial interest as protein-degrading additives to detergents. These reasons combined with their large data base make subtilisins attractive for protein engineering. Extensive engineering studies have been carried out on the Bacillus subtilins and more than 500 site-directed mutants have been produced to alter specific enzyme properties, such as pH profile, thermal stability or substrate specificity (see e. g. references[37 391). 

In contrast to the equilibrium-controlled approach the peptidase-catalyzed kinetically controlled peptide synthesis (for a review see reference1851) needs much less enzyme, the reaction time to reach maximal product yield is significantly shorter, and the product yield depends both on the properties of the enzyme used and the substrate specificity. Kinetic control means that the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. Whereas the equilibrium-controlled approach ends with a true equilibrium, in the kinetic approach the concentration of the product formed goes through a maximum before the slower hydrolysis of the product becomes important. The product will be hydrolyzed if the reaction is not stopped after the acyl donor ester is consumed and true equilibrium is allowed to be reached. 

The signal sequence is proteoly tically cleaved by signal pepetidase (member enzyme of translocon) once protein is translocated. The eukaryotic signal peptidases have multiple catalytic subunits with broad substrate specificity. The enzyme generally cleaves at a site that has small aliphatic residues at position -1 and -3. On-line prediction of signal peptide and cleavage site is available at PrediSi (http //www.predisi.de). 

It is interesting to note that there is another carboxy-peptidase. carboxypeptidase B (CPB), in the same tissue that has an ainino acid sequence homologous to that of CPA but differs in substrate specificity. Whereas CPA prefers aromatic (phenylalanine, tyrosine, and tryptophan) or hydrophobic amino acid residues (valine, leucine, and... 

The nomenclature of this large group of enzymes is difficult because the overall reaction of all peptidases and proteinases is essentially the same and substrate specificity in the ordinary sense is lacking. Many proteases attack nearly all denatured proteins and many native proteins. The two basic groups of these enzymes are the peptidases (expopeptidases 3.4.11-17) and the proteinases... 

Aertgeerts K, Ye S, Tennant MG, Kraus ML, Rogers J, Sang B-C, Skene RJ, Webb DR, Prasad GS (2004) Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation. Protein Sci 13 412-421... 

The substrate specificity of enzymes shows the following differences. The occurrence of a distinct functional group in the substrate is the only prerequisite for a few enzymes, such as some hydrolases. This is exenqtlified by nonspecific lipases (cf. Table 3.21) or peptidases (cf. 1.4.5.2.1) which generally act on an ester or peptide covalent bond. 

The human organism is not able to use dietary proteins as such. They must be hydrolysed into single amino acid molecules before they can be absorbed. The hydrolysis of proteins (mostly denatured proteins) is catalysed by proteolytic enzymes called proteases (proteinases or peptidases), which have relatively high substrate specificity. They catalyse the hydrolysis of interior peptide bonds to form peptides of different sizes (endopeptidases such as pepsin, trypsin and chymotrypsin) or attack the terminal amino acids (exopeptidases). Hydrolysis of the N-terminal amino acids is... 

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