Peptidases bond specificity

Table 1.2 Bond specificity of brush border peptidases Enzyme Bond hydrolysed...

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. 

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

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... 

Identification of carboxy-terminal amino acids was also attempted. Studies by Bergmann and his associates in the 1930s (see below) had characterized various peptidases with differing specificities. One of these was carboxypeptidase which required a free carboxy terminus adjacent to the peptide bond to be hydrolyzed. The specificity of the enzyme was limited but Lens in 1949 reported alanine to be at one end of insulin. Fromageot and his colleagues (1950) and Chibnall and Rees (1951) reduced the carboxy termini to B-aminoalcohols and showed glycine as well as alanine to be carboxy-terminal. Hydrazinolysis was also attempted the dry protein was treated with hydrazine at 100 °C for 6 h so that the carboxy-terminal amino acid was released as the free... 

The mechanism of hydrolysis of cysteine peptidases, in particular cysteine endopeptidases (EC 3.4.22), shows similarities and differences with that of serine peptidases [2] [3a] [55 - 59]. Cysteine peptidases also form a covalent, ac-ylated intermediate, but here the attacking nucleophile is the SH group of a cysteine residue, or, rather, the deprotonated thiolate group. Like in serine hydrolases, the imidazole ring of a histidine residue activates the nucleophile, but there is a major difference, since here proton abstraction does not appear to be concerted with nucleophilic substitution but with formation of the stable thiolate-imidazolium ion pair. Presumably as a result of this specific activation of the nucleophile, a H-bond acceptor group like Glu or Asp as found in serine hydrolases is seldom present to complete a catalytic triad. For this reason, cysteine endopeptidases are considered to possess a catalytic dyad (i.e., Cys-S plus H-His+). The active site also contains an oxyanion hole where the terminal NH2 group of a glutamine residue plays a major role. 

This enzyme [EC 3.4.16.5] (also known as serine-type carboxypeptidase I, cathepsin A, carboxypeptidase Y, and lysosomal protective protein) is a member of the peptidase family SIO and catalyzes the hydrolysis of the peptide bond, with broad specificity, located at the C-terminus of a polypeptide. The pH optimum ranges from 4.5 to 6.0. The enzyme is irreversibly inhibited by diisopropyl fluorophosphate and is sensitive to thiolblocking reagents. 

This lysosomal enzyme [EC 3.4.22.1], also known as cathepsin Bl, is a member of the peptidase family Cl. The catalyzed reaction is the hydrolysis of peptide binds with a broad specificity. The enzyme prefers the ArgArg—Xaa bond in small peptide substrates (thus distinguishing this enzyme from cathepsin L). The enzyme also exhibits a peptidyl-dipeptidase activity, releasing C-terminal dipeptides from larger polypeptides. 

This lysosomal endopeptidase [EC 3.4.23.5] is similar to pepsin A, except that the specificity is narrower and will not hydrolyze the Gln" —His peptide bond in the B chain of insulin. The enzyme is a member of the peptidase family Al. 

Glutamyl endopeptidase [EC 3.4.21.19] (also known as staphylococcal serine proteinase, V8 proteinase, protease V8, and endoproteinase Glu-C), a member of the peptidase family S2B, catalyzes the hydrolysis of Asp-Xaa and Glu-Xaa peptide bonds. In appropriate buffers, the specificity of the bond cleavage is restricted to Glu-Xaa. Peptide bonds involving bulky side chains of hydrophobic aminoacyl residues are hydrolyzed at a lower rate. 

Glutamyl endopeptidase 11 [EC 3.4.21.82], also known as glutamic acid-specific protease, catalyzes the hydrolysis of peptide bonds, exhibiting a preference for Glu-Xaa bonds much more than for Asp-Xaa bonds. The enzyme has a preference for prolyl or leucyl residues at P2 and phenylalanyl at P3. Hydrolysis of Glu-Pro and Asp-Pro bonds is slow. This endopeptidase is a member of the peptidase family S2A. 

This endopeptidase [EC 3.4.22.2], a member of the Cl peptidase family hydrolyzes peptide bonds in proteins, exhibiting a broad specificity for those bonds. There is a preference for an amino acyl residue bearing a large hydrophobic side chain at the P2 position and the enzyme does not accept a valyl residue at Pi. 

This manganese-dependent enzyme [EC 3.4.11.5] catalyzes the release of an N-terminal prolyl residue from a peptide. The mammalian enzyme, which is not specific for prolyl bonds, is possibly identical with cytosol amino-peptidase [EC 3.4.11.1]. 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

All peptidases catalyze the general reaction depicted in Figure C2.2.2, the hydrolysis of a peptide bond. The different peptidases are unique with respect to their specificity that is, their ability to accommodate particular sets of amino acids in the vicinity of a potentially scissile peptide bond. Some peptidases have very broad specificities, such as papain, which has few limi-... 

Peptidases are often classified as either exopeptidases or endopeptidases, depending on the positional specificity of the bonds they hydrolyze. Exopeptidases act at peptide bonds located at either the N or C terminus of the protein. Those acting at the C terminus are referred to as carboxypeptidases, those acting at the N terminus as aminopeptidases. Endopeptidases, on the other hand, act at peptide bonds internal to the polypeptide chain. 

Hydrolytic reactions are catalyzed by extracellular hydrolases and mineral surfaces (Chrost, 1990 Hoffman, 1990). For enzymatic reactions, a defined substrate or moiety must match the catalytic site of a specific enzyme. The most studied examples in aquatic systems are glycosidases, peptidases, and phosphatases (Munster and De Hann, 1998 see Chapter 13). In general, hydrolytic reactions break the relatively labile C—N and C—O bonds that link monomers, generating lower molecular weight products more suitable for microbial consumption. 

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... 

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