Leucine substrate specificity

An entirely different property of subtilisin was affected by substituting leucine at the 222 location. Native BPN is extremely sensitive to the presence of oxidation agents, showing rapid inactivation when incubated in the presence of 0.3% H2O2 (Figure 4). The Leu-222 variant, in contrast, was found to be totally stable under the same oxidation conditions. The data clearly show that single amino acid alterations can have dramatic effects upon the activity of the enzyme. Similarly, other changes have been shown to affect catalytic properties, substrate specificities and thermostability (7,2,9). 

Aminopeptidases that catalyze the hydrolysis of cysteinyl peptides are known. The membrane-bound aminopeptidases are glycoproteins, usually with molecular weights of about 100,000 daltons. They appear to be metalloproteins, one of the better known being a zinc-containing enzyme. Other enzymes, such as the leucine aminopeptidase, are cytosolic but, at least in this case, are also zinc-containing. The substrate specificity of these enzymes varies but most are relatively nonspecific. 

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... 

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. 

More recently, the substrate specificity of /9-secretase has been explored and compared with that of other aspartic proteases using a range of dodecameric substrates based mainly on the j3 -cleavage site of APP (67). The substrate recognition site of /3-secretase extended over several amino acids, and /9-secretase accepted a wide range of peptidic substrates. In common with other aspartic proteases, /9-secretase prefers a leucine residue at position PI. However, unlike these enzymes, /3-sccrctasc accepts polar or even acidic residues at positions PI and P2. and prefers bulky hydrophobic residues, preferably valine, at position P3. 

The substrate specificity of LeuDHs, catalyzing mainly branched-chain a-keto acids to the a-amino acids, has been investigated by Zink and Sanwal (1962) 36) and subsequently by Schiitte et al. (1985 B. cereus) t6, Ohshima and Soda (1989 Bacillus stearothermophilus and Bacillus sphaericus)1S1, Nagata et al. (1990 Bacillus DSM 7330) l37 Misono et al. (1990 Corynebacterium pseudodiphtheriticum) 38 and by Bommarius et al. (1994 Bacillus stearothermophilus)l39. In addition to the proteino-genic amino acids valine, leucine, and isoleucine, unnatural amino acids such as tert-leucine1401 or L-(3-hydroxy-valine[411 can be synthesized. 

Amino acid racemase with low substrate specificity catalyzes racemization of leucine and various other amino acids, which are also a-deuterated in 2H20 during their racemization[63). Therefore, [4S-2H]-NADH was produced in the same manner as described above with the racemase and L-leucine dehydrogenase (E. C. 1.4.1.9), which is pro-S specific[35). 

In the oxidative deamination reaction, the enzyme was active toward N-[l-D-(carboxyl)ethyl]-L-methionine, N-[l-D-(carboxyl)ethyl]-L-phenylalanine, etc. The substrate specificity for amino donors of ODH in the reductive secondary amine-forming reaction was examined with pyruvate as a fixed amino acceptor [15,24]. The enzyme utilized L-norvaline, L-2-aminobutyric acid, L-norleucine, P-chloro-L-alanine, o-acetyl-L-serine, L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-homophenylalanine, L-leucine, L-alanine, etc. 3-Aminobutyric acid and L-phenylalaninol also acted as substrates for the enzyme. Other amino compounds, such as P-amino acids, amino acid esters and amides, amino alcohols, organic amines, hydroxylamines, and hydrazines, were inactive as substrates. Pyruvate, oxaloacetate, glyoxylate, and a-ketobutyrate were good amino acceptors. We named the enzyme as opine... 

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