Endopeptidases specificity

Schiavo, G., Shone, C.C., Rossetto, O., Alexander, F.C. and Montecucco, C., Botulinum neurotoxin serotype 1 is a zinc endopeptidase specific for VAMP/ synaptobrevin, J. Biol. Chern., 268, 11516-11519, 1993. 

Several different proteases can attack a single protein at enzyme-selective amino-acid sequences. Proteases can be divided into two categories. Endopeptidases are enzymes that cleave peptide bonds between specific, nonterminal amino acids. There are endopeptidases specific for just about every amino acid. Exopeptidases are enzymes that cleave terminal peptide bonds at either the C-terminus or N-terminus. 

Schiavo G, Poulain B, Rossetto O, Benfenati F, Tauc L, Montecucco C (1992 b) Tetanus toxin is a zinc protein and its inhibition of neurotrasmitter release and protease activity depend on zinc. In EMBOJ. 11 3577-83 Schiavo G, Rossetto O, Santucci A, DasGupta BR, Montecucco C (1992 c) Botulinum neurotoxins are zinc proteins. In J. Biol. Chem. 267 23479-83 Schiavo G, Rossetto O, Catsicas S, Polverino de Laureto P, DasGupta BR, Benfenati F, Montecucco C (1993 a) Identification of the nerve-terminal targets of botulinum neurotoxins serotypes A, D and E. In J. Biol. Chem. 268 23784-7 Schiavo G, Santucci A, DasGupta BR, Metha PP, Jontes J, Benfenati F, Wilson MC, Montecucco C (1993 b) Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. In FEBS Lett. 335 99-103 Schiavo G, Shone CC, Rossetto O, Alexandre FCG, Montecucco C (1993 c) Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synOp-tobrevin. In J. Biol. Chem. 268 11516-9... 

Elastase (EC 3.4.21.11) an endopeptidase specific for the Elastic (see) in animal elastic fibers. Its inactive precursor, proelastase, is formed in the vertebrate pancreas and converted in the duodenum to elastase by the action of trypsin. The natural substrate of E. is elas-tin, an insoluble protein rich in valine, leucine and isoleucine. E. attacks the peptide bond adjacent to a nonaromatic, hydrophobic amino acid. The best synthetic substrates are therefore acetyl-Ala-Ala-Ala-OCHj and benzoylalanine methyl ester. Benzoylarginine ester (a trypsin substrate), and acetyltyrosine ester (a chymotrypsin substrate) are not attacked by E. 

The most ingenious exocytosis toxins, however, come from the anaerobic bacteria Clostridium botulinum and Clostridium tetani. The former produces the seven botulinum neurotoxins (BoNTs) A-G the latter produces tetanus neurotoxin (TeNT). All eight toxins consist of a heavy (H) chain and a light (L) chain that are associated by an interchain S-S bond. The L-chains enter the cytosol of axon terminals. Importantly, BoNT L-chains mainly enter peripheral cholinergic terminals, whereas the TeNT L-chain mainly enters cerebral and spinal cord GABAergic and glycinergic terminals. The L-chains are the active domains of the toxins. They are zinc-endopeptidases and specifically split the three core proteins of exocytosis, i.e. the SNAREs (Fig. 1 inset). Each ofthe eight toxins splits a... 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

Proteases (endopeptidases or proteinases) commonly used for specific cleavage of proteins are summarised in Table 6.2. Trypsin is almost always used as an enzyme of first choice it is highly specific and stable, has an appropriate pH-optimum and is commercially available in high purity and quality. When the results obtained are ambiguous, or the trypsin cannot be used for any other reason, a different protease can be easily chosen. In all experiments, described here, the trypsin cleavage was applied. 

E. Theodoratou, A. Paschos, S. Mintz-Weber, A. Bock (2000) Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli. Arch. Microbiol., 173 110-116... 

One of the general principles of the Nomenclature Committee is that enzymes should be classified and named according to the reaction they catalyze. However, the overlapping specificities of and great similarities in the action of different peptidases render naming solely on the basis of function impossible [10]. For example, some enzymes can act as both endo- and exopeptidases. Thus, cathepsin H (EC 3.4.22.16) is not only an endopeptidase but also acts as an aminopeptidase (EC 3.4.11), and cathepsin B (EC 3.4.22.1) acts as an endopeptidase as well as a peptidyl-dipeptidase (EC 3.4.15). The actual classification of peptidases is, therefore, a compromise based not only on the reaction catalyzed but also on the chemical nature of the catalytic site, on physiological function, and on historical priority. 

The evolutionary classification has a rational basis, since, to date, the catalytic mechanisms for most peptidases have been established, and the elucidation of their amino acid sequences is progressing rapidly. This classification has the major advantage of fitting well with the catalytic types, but allows no prediction about the types of reaction being catalyzed. For example, some families contain endo- and exopeptidases, e.g., SB-S8, SC-S9 and CA-Cl. Other families exhibit a single type of specificity, e.g., all families in clan MB are endopeptidases, family MC-M14 is almost exclusively composed of carboxypeptidases, and family MF-M17 is composed of aminopeptidases. Furthermore, the same enzyme specificity can sometimes be found in more than one family, e.g., D-Ala-D-Ala carboxypeptidases are found in four different families (SE-S11, SE-S12, SE-S13, and MD-M15). 

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. 

The hydrogenase specific endopeptidases (HybD, HyaD, Hyl, HoxM, HupM, HupD)... 

The proteolytic enzymes are classified into endopeptidases and exopeptidases, according to their site of attack in the substrate molecule. The endopeptidases or proteinases cleave peptide bonds inside peptide chains. They recognize and bind to short sections of the substrate s sequence, and then hydrolyze bonds between particular amino acid residues in a relatively specific way (see p. 94). The proteinases are classified according to their reaction mechanism. In serine proteinases, for example (see C), a serine residue in the enzyme is important for catalysis, while in cysteine proteinases, it is a cysteine residue, and so on. 

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. 

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 3.4.22.15] is an lysosomal endopeptidase with specificity akin to papain. Cathepsin L displays a higher activity toward protein substrates than does cathepsin B. 

This enzyme [EC 3.4.22.6], also known as papaya proteinase II, is a member of the peptidase family Cl. It is the major endopeptidase of papaya (Carica papaya) latex. It has a specificity similar to that of papain. In addition, there are a number of chromatographic forms of the enzyme. 

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. 

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