Peptide protease-catalyzed

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

S. A. Bizzozero, H. Dutler, Stereochemical Aspects of Peptide Hydrolysis Catalyzed by Serine Proteases of the Chymotrypsin Type , Bioorg. Chem. 1981, 10, 46 - 62 ... 

W. Kullmann, Kinetics of Chymotrypsin- and Papain-Catalysed Synthesis of (Leu-cine)enkephalin and (Methionine)enkephahn , Biochem. J. 1984, 220, 405 - 416 W. Kullmann, Protease-Catalyzed Peptide Synthesis , Adv. Biosci. 1987, 65, 135- 140. 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Swann, P. G. Casanova, R. A. Desai, A. Frauenhoff, M. M. Urbancic, M. Slomczynska, U. Hopfmger, A. J. Le Breton, G. C. Venton, D. L. Nonspecific protease-catalyzed hydrolysis/synthesis of a mixture of peptides Product diversity and ligand amplification by a molecular trap. Biopolymers 1997, 40, 617-625. 

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. 

An alternative to the synthesis of proteins by classical fragment synthesis in solution or by solid-phase synthesis on a support is the use of enzyme-catalyzed condensation of amino acids or peptides. This possibility was first demonstrated in 1938 91 with the synthesis of poorly soluble benzoyl-leucyl-leucine anilide by papain catalysis. After many years, this approach was extended to the preparation of peptide hormones such as Leu-enkephalin 92 and dynorphin(l -8).[93 This was made possible by the use of highly purified enzymes and by careful control of reaction conditions. The basic principles of protease-catalyzed peptide bond formation have been discussed.194 ... 

Celovsky, V. and Bordusa, F. (2000). Protease-catalyzed fragment condensation via substrate mimetic strategy a useful combination of solid-phase peptide synthesis with enzymatic methods. /. Pept. Res., 55, 325-9. 

Jakubke, H.-D., Kuhl, P, and Konnecke, A. (1985). Basic principles of protease-catalyzed peptide bond formation. Angew. Chem. Int. Ed. Engl, 24, 85-93. 

Tholey, A., Zabet-Moghaddam, M. and Heinzle, E., Quantification of peptides for the monitoring of protease-catalyzed reactions by matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrixes. Anal. Chem., 78,291, 2006. 

The protease-catalyzed synthesis of peptide bonds is known as the plastein reaction ( ). Plastein itself is defined as the product formed by this reaction which is insoluble in trichloroacetic acid solutions 6). The plastein reaction has been most extensively investigated by researchers in Japan (, ... 

Cleaving the Polypeptide Chain Several methods can be used for fragmenting the polypeptide chain. Enzymes called proteases catalyze the hydrolytic cleavage of peptide bonds. Some proteases cleave only the peptide bond adjacent to particular amino acid residues (Table 3-7) and thus fragment a polypeptide chain in a predictable and reproducible way. A number of chemical reagents also cleave the peptide bond adjacent to specific residues. 

The stereochemical aspects of peptide hydrolysis catalyzed by chymotrypsin and related serine proteases has been recently analyzed with respect to requirements for stereoelectronic control of bond cleavage and this analysis has led to a much more complete understanding of the reaction mechanism (9-14). 

Schuster M, Aaviksaar A, Haga M, Ullmann U, Jakubke HD. Protease-catalyzed peptide synthesis in frozen aqueous systems the freeze concentration model. Biomed Biochim Acta 1991 50 S84-S89. 

Stevenson, D.E., Ofman, D.J., Morgan, K.R., and Stanley, R.A. 1998. Protease-catalyzed condensation of peptides as a potential means to reduce the bitter taste of hydrophobic peptides found in protein hydrolysates. Enzyme Microb. Technol. 22, 100-110. 

Synthetic peptide inhibitors have been developed for a variety of proteases [199-204]. Peptide inhibitors of the metalloprotease angiotensin I converting enzyme (ACE) are of major importance as hypertensive agents [13, 31]. A variety of peptides derived from protease-catalyzed hydrolysis of com cc-zein [202-203] or of wheat germ protein [199, 204] inhibit ACE (Table 6). The most potent of such plant-derived ACE inhibitory peptides is Ile-Val-Tyr (IVY) (Ki 0.1 xM) [199, 204], Further plant-derived peptide ACE inhibitors include the tripeptide glutathione [73, 82], the glutathione -related peptide Y-L-glutamyl-(+)-allyl-L-cysteine sulphoxide [73, 82, 200, 201] and the tripeptide His-His-Leu (HHL) from fermented soybean [201] (Table 6). 

Pepsin and the pancreatic proteases catalyze the conversion of dietary protein to peptides and amino acids. The aminopeptidases and the dipeptidases in the intestinal mucosa almost complete the hydrolysis of the peptides to amino acids, but some peptides, especially those containing glutamate, pass into the gut mucosal cells with the free amino acids. The aminopeptidases remove amino acids from the N-terminus of a peptide. 

Lehmann, W. D. (1996). Protease-catalyzed incorporation of 180 into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ ionization mass spectrometry. Electrophoresis 17, 945-953. 

In general the thiol proteases catalyze the hydrolysis of a variety of peptide, ester, and amide bonds of synthetic substrates. Employing the general formula R —NH—CHR—CO—X, cleavage of the —CO—X— bond has been demonstrated when R represents the side chain of glycine, threonine, methionine, lysine, arginine, citrulline, leucine, and tyrosine. 

In contrast to the equilibrium-controlled approach which ends with a true equUibrium, in the protease-catalyzed kinetically controlled synthesisf l the product appearing with the highest rate and disappearing with the lowest velocity would accumulate. This approach requires the use of acyl donor esters as carboxy components (Ac-X) and is limited to proteases which rapidly form an acyl-enzyme intermediate (Ac-E). Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophihc amino components via an acyl-enzyme intermediate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester, Ac-X, to form a covalent acyl-enzyme intermediate, Ac-E, that reacts, in competition with water, with the amino acid or peptide-derived nucleophile HN to form a new peptide bond (Scheme 3). The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control, and if k4[HN] k3[H20], the peptide product Ac-N should accumulate. However, the soluble peptide product will be degraded if the reaction is not terminated after the acyl donor ester is consumed. 

Table 1 Medium Engineering in Protease-Catalyzed Peptide SynthesisI " ...

In principle, the use of amino acid or peptide esters as nucleophilic components in protease-catalyzed synthesis is possible, but with a drastically decreased efficiency. However, acyl transfer to arginine or lysine alkyl esters in ice using a-chymotrypsin with regard to its strong preference for basic residues in the P/ position enabled synthesis of a N-protected tripeptide ester in high yield (Scheme 7, see Section 4.2.1.2.2). Furthermore, it was found to be the method of choice in synthesizing new potential protease substrates (for proteases with a preference for basic residues in the Pj position). Neither enzymatic synthesis at room temperature nor synthesis in organic solvents has been shown to proceed in a successful manner. 

Based on an extensive theoretical treatment of equilibrium positions,P l an interesting approach of solid-to-solid conversion catalyzed by proteases has been developed. According to this, a favorable equilibrium shift toward the peptide product is given when the starting reactants are largely undissolved in the reaction medium and the product precipitates. The use of solid-phase substrate pools combines the equimolar or nearly equimolar supply of reactants with high obtainable yields, easy workup procedures and, in principle, compatibility with conventional chemical peptide synthesis standard procedures. Both the advantage of solid-phase substrate pools mainly in equilibrium-controlled synthesisl " and the extension of this approach to protease-catalyzed acyl-transfer reactionst have been successfully demonstrated. 

Scheme 14 General Course of the Combined Solid-Phase Peptide Synthesis Substrate Mimetic Fragment Condensation Approach Generation of the Protected Peptide Fragment by Aminolysis Using an Amino Acid Substrate Mimetic, and Irreversible Protease-Catalyzed Fragment Condensation ...

In these protease-catalyzed cleavages of the C-terminal protecting groups, it has to be taken into consideration that an undesired hydrolysis of peptide bonds can occur, especially if unnatural and poor substrates are subjected to enzyme-mediated transformations. The use of enzymes which cannot cleave amides at all enables this undesired side reaction to be overcome. This principle has been realized in the development of the heptyl (Hep) ester [13,14,48,49] as carboxyl protecting group, which can be enzymatically removed by means of lipases (Fig. 10). 

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