Amino Acid-Specific Peptidases

Few microbial proteases acting on n-peptides are known. The alkaline D-peptidase (ADP) from Bacillus cereus is related to Du-carboxypeptidase and p-lactamases. These enz unes have an accessible groove in which the nucleophilic serine and other catalytic amino acids are located. This n-peptidase could be applied for the synthesis of the 92-amino acid peptidyl prolyl cis-trans isomerase from Escherichia coli by condensation of two peptide fragments, of which the 35-amino acid acyl donor was activated as the OGp ester [62]. Thus the D-amino acid-selective enzyme was used for preparing a protein composed of L-amino acids and making the product insensitive to hydrolysis by the coupling enzyme. 

15 DISCOVERY AND ENGINEERING OF ENZYMES FOR PEPTIDE SYNTHESIS AND ACTIVATION 

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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 introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

Insulin was first identified as an anti-diabetic factor in 1921, and was introduced clinically the following year. Its complete amino acid sequence was determined in 1951. Although mature insulin is a dimeric structure, it is synthesized as a single polypeptide precursor, i.e. preproinsulin. This 108 amino acid polypeptide contains a 23 amino acid signal sequence at its amino terminal end. This guides it through the endoplasmic reticulum membrane, where the signal sequence is removed by a specific peptidase. 

Amino-, carboxy- and endo peptidase which cleave peptides at specific amino acid linkages... 

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 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). 

After intravenous administration to rabbits, the t-amino acid derivatives of dapsone were rapidly (f1/2<2 min) and quantitatively converted to dapsone. The corresponding D-amino acid derivatives were also quantitatively converted to dapsone, but the f1/2 values ranged from 30 to 60 min. In human blood, the f1/2 values for hydrolysis of the L-amino acid prodrugs ranged from 1.7 to 20 min (Leuleucine peptidase, with decreasing specificity constants (kcat/Km) Leu=Ala>Phe>Lys=Gly. Interestingly, only L-Lys-dapsone was a substrate of trypsin, whereas only L-Phe-dapsone was a substrate of chymotrypsin. 

AM-001, and mannanase B properties are similar to those of P-mannanase M-III. Furthermore, the Ouchterlony double diffusion test showed that these five enzymes gave fused precipitation lines. However, N-terminal amino acid sequences of the five mannanases determined by an automatic amino acid sequencer revealed that the N-terminal amino acid sequence from amino acid 1 (Asn) to 9 (Gin) of the Bacillus sp. AM-001 enzymes coincides with those from amino acid 4 (Asn) to 12 (Asn) of the R coll JMlOl (pMAH3) enzymes as shown in Fig. 4. This may reflect differences in the specificities of the signal peptidases of the two bacteria. 

Combinations of several enzymes with different specificities are required for complete degradation of proteins into free amino acids. Proteinases and peptidases are found not only in the gastrointestinal tract (see p. 268), but also inside the cell (see below). 

The starter cells begin to die off at the end of curd manufacture (Figure 10.21) the dead cells may lyse and release their intracellular endopeptidases (Pep O, Pep F), aminopeptidases (including Pep N, Pep A, Pep C, Pep X), tripeptidases and dipeptidases (including proline-specific peptidases) which produce a range of free amino acids (Figure 10.22). About 150 peptides have... 

A distinct group of synthetic depsipeptides comprises of compounds that do not originate from natural product biodiversity several artificial substrates of peptidases and esterases belong to this group, as well as polydepsipeptides that are considered as potentially biodegradable polymeric materials. A specific feature of depsipeptide synthesis is the necessity to acylate a hydroxy acid component, which requires stronger activation of the amino acid component in comparison to normal peptide synthesis. Otherwise, the main principles of depsipeptide synthesis are similar to those of peptides. Frequently, formation of the ester... 

Carboxypeptidase A catalyzes the hydrolysis of carboxyl-terminal acidic or neutral amino acids however, the rate of hydrolysis depends on the structure of the side chain R. Amino acids with nonpolar aryl or alkyl side chains are cleaved more rapidly. Carboxypeptidase B is specific for the hydrolysis of basic COOH-terminal amino acids (lysine and arginine). Neither peptidase functions if proline occupies the COOH-terminal position or is the next to last amino acid. 

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-... 

There are at least three peptidases in the brush border of the small intestine Aminopeptidase A, which has an affinity for peptide-bound acid amino acids (11), aminopeptidase N, which has a broad specificity (12), and dipeptidylpeptidase IV, which releases dipeptides from the N-terminal end of peptides with a preference for X-PRO terminals (13). In Table III are shown the effect of a low concentration ofThe LMW fraction on the activity of these enzymes in extracts of hog intestine. Aminopeptidase N was found to be strongly inhibited by 0.25 mg/ml of the fraction. Aminopeptidase A and dipeptidylpeptidase IV were not inhibited. 

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