Glu-specific protease

Scheme 5.1.2. Structures of substrates and substrate mimetic moieties for Arg- and Glu-specific proteases. 1. arginine 2. 4-amidinophenyl ester 3. 4-guanidinophenyl ester (OGp) 4. aspartic acid 5. glutamic acid 6. carboxymethyl thioester (SCm).

Lycopersicon esculentum (tomato) (Solanaceae) [leaf] ERI(71aa 8kDa 2Cys lS-S) Glu-specific protease (E47-D48) [462]... 

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. 

PeptideMass (ExPASy Proteomic tools), which is designed for use in peptide mapping experiments, determines the cleavage products of a protein after exposure to a specific protease or chemical reagent. The enzymes and reagents available for cleavage via PeptideMass are trypsin, chymotrypsin, Lys C, cyanogen bromide, Arg C, Asp N, and Glu C. 

As exemplified above, a ligation needs an N-terminal Cys and a C-terminal thioester as prerequisites. In chemical synthesis these two conditions are obviously easily achieved. However, recombinant proteins are synthesized with an N-terminal Met and the C-terminus is just a free carboxyl group. There are various methods available to generate an N-terminal Cys. In a commonly used strategy a specific protease cleaving site is introduced. The Tobacco Etch Virus (TEV) protease recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves the... 

In rapid turnover the cleavage site by a proposed specific protease was closed in by Edelman et al. to be at the sequence around glu 242 (19). A PEST sequence was proposed to occur on the N-side of arg 238 (19). It is long known that the cleavage is influenced by herbicides in the Qg pocket (24). 

Wehofsky, N., Bordusa, F. (1999). Programming of enzyme specificity by substrate mimetics investigations on the Glu-specific V8 protease reveals a novel general principle of biocatalysis. FEBS Lett., 443,220-224. 

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. 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

While trypsin cuts the peptide linkages Lys-X and Arg-X, a fungal protease cleaves only X-Lys.227 A protease from the submaxillary glands of mice cleaves only Arg-X,228 one from Staphylococcus specifically at Glu-X,229/230 and one from kidneys at Pro-X.231... 

Among cysteine proteases of bacteria is a papainlike enzyme from Clostridium histolyticum with a specificity similar to that of trypsin 338 Tire anaerobic Porphyromonas gingivalis, which is implicated in perio-dental disease, produces both arginine- and lysine-specific cysteine proteases designated gingipains.339 3393 Some virally encoded cysteine proteases, including one from the polio virus, have trypsin-like sequences with the serine of the catalytic triad replaced by cysteine.340 341 A human adenovirus protease also has a Cys His Glu triad but a totally different protein fold.342... 

Both alkaline proteases form an intermediate, the acyl-enzyme complex, on the reaction coordinate from the amino acid component to the dipeptide, which is formed by the triad Ser-(or Cys-)-His-Asp (or -Glu) (see Chapter 9, Section 9.5). The acyl-enzyme complex can be formed with the help of an activated amino acid component such as an amino acid ester. The complex can react either with water to the undesired hydrolysis product, the free amino acid, or with the amine of the nucleophile, such as an amino acid ester or amide, to the desired dipeptide. The particular advantage of enzyme-catalyzed peptide synthesis rests in the biocatalyst specificity with respect to particular amino acids in electrophile and nucleophile positions. Figure 7.26 illustrates the principle of kinetically and thermodynamically controlled peptide synthesis while Table 7.3 elucidates the specificity of some common proteases. 

In order to generate two different peptide series, the protein is digested with trypsin, which specifically cleaves on the C-terminal side of Lys and Arg, and by protease V8, which specifically cleaves on the C-terminal side of Glu and Asp (see Table 8.4). 

Thermitase, a thermostable extracellular serine protease from the thermophrhc microorganism Thermoactinomyces vulgaris, with an esterase/protease activity ratio >1000 1 shows a broad amino acid side-chain tolerance and cleaves methyl, ethyl, benzyl, ethox-ybenzyl, and ferf-butyl esters from a variety of Nps-, Boc-, Bpoc-, and Z-protected di- and oligopeptides in high yields at pH 8 and 33-55 °C (Scheme 15 ).[28,29,60-62] jjj addition, it is specific for the a-carboxy groups of Asp and Glu. 

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