Phenylalanine substrate specificity

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. 

Drosophila DDC belongs to a family of pyridoxal-dependent decarboxylases that extends from prokaryotes to eukaryotic plants and animals. The members of this family show significant sequence similarity over much of their length, even though the individual proteins have quite different substrate specificities, including the amino acids tyrosine, tryptophan, phenylalanine, histidine, and glutamate, and the amino acid derivatives... 

We have used a series of biocatalysts produced by site-directed mutations at the active site of L-phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus, which expand the substrate specificity range beyond that of the wild-type enzyme, to catalyse oxidoreduc-tions involving various non-natural L-amino acids. These may be produced by enantiose-lective enzyme-catalysed reductive amination of the corresponding 2-oxoacid. Since the reaction is reversible, these biocatalysts may also be used to effect a kinetic resolution of a D,L racemic mixture. ... 

Based on the known substrate specificity of a-chymotrypsin, phenylalanine has been chosen as the amino acid at the Pi position (P-nomenclature according to Schechter and Berger) [55]. The a-proton at Pi has been substituted either by methyl or trifluoromethyl. Substitutions beyond Pi contain trifluoromethyl alanine or aminoisobutyric acid. Therefore, each fluorosubstitution can be compared to its natural as well as fluorine-free a-substituted analog, thereby enabling differentiation of the steric and electronic effects. Scheme 2 summarizes the amino acids that have been used in this study. 

In this transamination, the effect of para substitient groups has been studied using fluorinated phenylpyruvic acids and L-aspartic acid. From these results, the migratory preference is H > F > Cl > Br > CF3. This order has been attributed to the bulkiness of the substituted group [57]. Direct amination of p-substituted succinic acid with phenylalanine ammonialyase (EC 4.3.1.5) has suggested very high substrate specificity that the order of reaction rate is m-F o-F P-p-F >CF3. 

The whole question of the specificity was reopened with the discovery that E. coli phosphatase, contrary to an earlier statement (114), hydrolyzed a variety of polyphosphates including metaphosphate of average chain length 8 (97). It was subsequently reported that partially purified phosphatases from several mammalian tissues had appreciable PPi-ase activity at pH 8.5 (115). This was confirmed (116) and extended to include ATPase and fluorophosphatase activities (117). Proof that the same enzyme is responsible for the monoesterase and PPi-ase activities was afforded by heat inactivation studies, cross inhibition experiments, and inhibition of PPi-ase activity by L-phenylalanine, a specific inhibitor of intestinal phosphatase. It was also found that calf intestinal phosphatase couid be phosphorylated by 32P-PP and the number of sites so labeled agreed with the number of active sites determined with a monoester substrate using a stopped-flow technique (118). It would seem that the main reason for the confusion with regard to the PPi-ase activity results from the inclusion of Mg2+ in the assay. This stimulates the monoesterase activity but almost completely inhibits PPi-ase activity (117). 

When switching from water to an organic solvent, or switching between organic solvents, the substrate specificity can change. In the example of the standard reaction, transesterification of N-acetyl-i-phenylalanine ethyl ester with n-propanol by Subtilisin Carlsberg, which has been mentioned several times in this chapter already, the relative specificity between the rather hydrophobic phenylalanine compound and its more hydrophilic analog N-acetyl-L-serine ethyl ester varies with the solvent (Table 12.8) (Wescott, 1993). 

Beauvericin is a structural homolog of enniatins in which the branched-chain L-amino acid is substituted by the aromatic amino acid L-phenylalanine. Beauvericin synthetase, which has been isolated from the fungus Beauveria bassiana [54] and various strains of Fusaria [55], strongly resembles Esyn with respect to its molecular size and the reaction mechanism. In contrast to Esyn, which is only able to incorporate aliphatic amino acids, beauvericin synthetase exhibits high substrate specificity for aromatic amino acids such as phenylalanine. This capability is obviously caused by mutational alterations in the adenylation domain of this enzyme. 

In the mid to late 1980s, many research groups focused on methods and processes to prepare L-phenylalanine (Chapter 3). This was a direct result of the demand for the synthetic, artificial sweetener aspartame. One of the many routes studied was the use of phenylalanine dH (Scheme 19.4, R = C6H5CH2) with phenylpyruvate (PPA) as substrate.57-58 This enzyme from Bacillus sphaericus shows a broad substrate specificity and, thus, has been used to prepare a number of derivatives of L-phenylalanine.59 A phenylalanine dH isolated from a Rhodococcus strain M4 has been used to make L-homophenylalanine (.S )-2-amino-4-pheny I butanoic acid], a key, chiral component in many angiotensin-converting enzyme (ACE) inhibitors.40 More recently, that same phenylalanine dH has been used to synthesize a number of other unnatural amino acids (UAAs) that do not contain an aromatic sidechain.43... 

Leucine dH is the enzyme used as the biocatalyst in the process commercialized by Degussa AG (Hanau, Germany) to produce L-tert-leucine (L-Tle).28-60 This UAA has found widespread use in peptidomimetic drugs in development, and the demand for this unique amino acid continues to increase.61-62 This process, which has been the subject of much study, requires a co-factor recycling system (Scheme 19.4, R = Me3C).63-64 Similar to phenylalanine dH, leucine dH has been used to prepare numerous UAAs because of its broad substrate specificity.43-65-66... 

Aspartase exhibits incredibly strict substrate specificity and thus is of little use in the preparation of L-aspartic acid analogues. However, a number of L-phenylalanine analogues have been prepared with various PAL enzymes from the yeast strains Rhodotorula graminis, Rhodotorula rubra, Rhodoturula glutinis, and several other sources that have been cloned into E. call.243 241 Future work in this area will likely include protein engineering to design new enzymes that offer a broader substrate specificity such that additional L-phenylalanine analogues could be prepared. 

Numerous other amino acid decarboxylases have been isolated and characterized, and much interest has been shown as a result of the irreversible nature of the reaction with the release of C02 as the thermodynamic driving force. Although these enzymes have narrow substrate-specificity profiles, their utility has been widely demonstrated. Additional industrial processes will continue to be developed once other decarboxylases become available. Such biocatalysts would include the aromatic amino acid (E.C. 4.1.1.28), phenylalanine (E.C. 4.1.1.53) and tyrosine (E.C. 4.1.1.25) decarboxylases, which likely could be used to produce derivatives of their respective substrates. These derivatives are finding increased use in the development of peptidomimetic drugs and as possible positron emission tomography imaging agents.267-268... 

An example of a project of this nature is the screening for microorganisms to produce aspartame (28) from a precursor that is easy synthesized by chemical methods. Microorganisms were screened for their ability to catalyze the trans-addition of ammonia across the double bond of /V-fumaryl-L-phenylalanine methyl ester (FumPM) (74) (Scheme 19.43).403 This is essentially the reaction of a mutated aspartase because the native enzyme has such strict substrate specificity (see Section 19.2.4). Although the literature touts this as a successful screening effort, this process has not been practiced commercially because the yields are extremely low.404405... 

These features of substrate specificity of tTG are mirrored by epitope specificity of gliadin antibodies of CD patients. The common core of the nona-peptides specifically recognized by antibodies of CD patients is QPEQPFP with the amino acid proline residing in position +2 [127]. However, in accordance with results from studies of substrate specificity of tTG [182], exchange of phenylalanine residue for tyrosine completely abolished binding of IgA of CD patients [127]. 

Studies of epitope specificity of gliadin antibodies and substrate specificity of tTG point to a role of proline and phenylalanine in amino acid positions +2 and +3 in glutamine deamidation. In contrast, investigations of T-cell stimulatory peptides reveal several potent T-cell stimulatory peptides from the N-terminal part of a-type gliadin containing the PELPY motif. In this connection, it was shown that substitution of phenylalanine in the +3 position or other amino acids in the glutenin peptide Git-156 inhibited the T-cell proliferative response [186]. 

Trypsin cleaves a peptide bond on the C-terminal side of a basic residue such as arginine (Arg) or lysine (Lys) whereas chymotrypsin cleaves on the C-terminal side of the hydrophobic residues phenylalanine (Phe), tryptophan (Trp) or tyrosine (Tyr). Elastase cleaves on the C-terminal side of small amino acids such as alanine (Ala) and glycine (Gly). A large number of serine PI proteins have been isolated from plants (Table 13.4) and the substrate specificity of the target proteases corresponds with the inhibitory amino acid sequences (P2-P1-PT-P2 ) of the PI proteins. Thus, the double-headed trypsin- and chymotrypsin-inhibitory Bowman-Birk PI protein 1 (BBI-1) from soybean (Glycine BBI-1, Table 13.5G) has a Pl-PT sequence of Lys—Ser at the trypsin inhibitory domain I site and a PI PI sequence of Leu-Ser at the chymotrypsin inhibitory domain II site. 

An illustrative example of a change in chemoselectivity is the inversion of substrate specificity of the serine protease Subtilisin Carlsberg in the transesterification reaction of ethyl esters of A-acetyl-L-serine and A-acetyl-L-phenylalanine with 1-propanol, measured in twenty anhydrous organic solvents. The enzyme-catalysed reaction with the serine substrate is strongly favoured in dichloromethane, while the reaction with the phenylalanine substrate is preferred in t-butylamine, with a 68-fold change in substrate specificity [313]. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

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