Leucine hydrolysis

Aminopeptidase, Leucine Hydrolysis of /V-terminal amino acid, which is preferably leucine, but may be other amino acids, from proteins and oligopeptides, yielding free amino acids and oligopeptides of lower molecular weight. 

Substrate Hydrolysis amino-terminal to leucine Hydrolysis carboxyl-terminal to leucine Bond not hydrolyzed ... 

Thraustomycin. Thraustomycin and P-thraustomycin are isolated from S. exfoliatus (4). Although their stmctures have not been totally elucidated, hydrolysis of thraustomycin shows that it contains equimolar quantities of adenine, L-leucine, and a tetrahydroxymonocarboxyhc acid. Thraustomycin is a potent inhibitor of the fungus, Af. hiemallis (+), but does not inhibit bacteria. 

Over 30 amino acids have been identified in the hydrolysis product of casein of which glutamic acid, hydroxyglutamic acid, proline, valine, leucine and lysine comprise about 60%. The residues of the aminoacid arginine also appear to be of importance in the cross-linking of casein with formaldehyde. 

The amino acid leucine is biosynthesized from n-ketoisocaproate, which is itself prepared from -ketoisovalerate by a multistep route that involves (1) reaction with acetyl CoA, (2) hydrolysis, (3) dehydration, (4) hydration. (5) oxidation, and (6) decarboxylation. Show lhe steps in the transformation, and propose a mechanism for each. 

On complete hydrolysis, a polypeptide gives two alanine, one leucine, one methionine, one phenylalanine, and one valine residue. Partial hydrolysis gives the following fragments Ala-Phe, Leu-Met, Val-Ala, Phe-Leu. It is known that the first amino acid in the sequence is valine and the last one is methionine. What is the complete sequence of amino acids ... 

A 1.00-mg sample of a pure protein yielded on hydrolysis 0.0165 mg of leucine and 0.0248 mg of isoleucine. What is the minimum possible molar mass of the protein (MM leucine = MM isoleucine = 131 g/mol)... 

The Jacobsen group has also shown that the recycling of the resin-bounded catalyst can be successfully performed [152,154]. Moreover, they have developed an efficient method for the hydrolysis of the aminonitrile into the corresponding amino acid. This method was apphed for the commercial production of optically active K-amino acids at Rhodia ChiRex (e.g. tert-leucine) the catalyst was immobihsed on a resin support (4 mol %, 10 cycles) and the intermediate hydrocyanation adduct was trapped by simply replacing TFAA with HCOOH/AC2O, for example. Highly crystalhne formamide derivatives were thus obtained in excellent yields (97-98% per cycle) with very high enantioselectivities (92-93% per cycle) [158]. 

Procarboxypeptidase A is activated by the removal of a peptide of some 64 residues from the N-terminus by trypsin.153 This zymogen has significant catalytic activity. As well as catalyzing the hydrolysis of small esters and peptides, procarboxypeptidase removes the C-terminal leucine from lysozyme only seven times more slowly than does carboxypeptidase. Also, the zymogen hydrolyzes Bz-Gly-L-Phe with kcsA = 3 s-1 and KM = 2.7 mM, compared with values of 120 s 1 and 1.9 mM for the reaction of the enzyme.154 In contrast to the situation in chymotrypsinogen, the binding site clearly pre-exists in procarboxypeptidase, and the catalytic apparatus must be nearly complete. 

Rapid monoalkylations are achieved in good yield compared with classical methods. Of particular interest is the synthesis of ot-amino acids by alkylation of aldimines with microwave activation. Subsequent acidic hydrolysis of the alkylated imine provides leucine, serine, or phenylalanine in preparatively useful yields within 1-5 min [50], Alkylation of phenylacetonitrile was performed by solid-liquid PTC in 1-3 min under microwave irradiation (Eq. 36 and Tab. 5.14). The nitriles obtained can subsequently be quickly hydrolyzed in a microwave oven to yield the corresponding amides or acids [56]. 

The amounts of single amino acids excreted in urine in the conjugated form, as determined independently by Stein and Muting, are given in Tables 1 and 2. According to Stein, glycine, glutamic acid, aspartic acid, histidine, and proline are quantitatively the most important amino acids liberated in the course of urine hydrolysis. Serine, lysine, tyrosine, cysteine and cystine, threonine, alanine, valine, phenylalanine, and leucine are... 

Stein et al. found in the course of experiments dealing with free and conjugated urinary amino acids in Wilson s disease (S9) that besides a marked aminoaciduria, almost a twofold increase in the excretion of all bound amino acids could be observed. As compared with normal urine (S8), unusual amounts of conjugated leucine, isoleucine, and valine are excreted in cases of Wilson s disease. Also the increase of glutamic acid, aspartic acid, and phenylalanine after urine hydrolysis is much more distinct in this disease than in normal conditions. Other bound amino acids are at or below normal levels. 

Micelle-forming copper complexes were found to effectively discriminate between enantiomers in the hydrolysis of a-amino esters (257). Hydrolysis of (.V)-phenylalanine p-nitrophenyl ester is 14-fold faster than its enantiomer, Eq. 223. Leucine affords 10-fold faster hydrolysis. The authors note that the micellar nature of these systems is extremely important for both rate of hydrolysis and selectivity (258). For example, the /V-mcthyl-dcrivcd ligand 419b leads to inhibition of the hydrolysis process, relative to catalysis by Cu(II) ion alone. 

See Section IV.1 for alternative methods of chiral resolution. Partial chemical hydrolysis of proteins and peptides with hot 6 M HC1, followed by enzymatic hydrolysis with pronase, leucine aminopeptidase and peptidyl D-amino acid hydrolase, avoids racemiza-tion of the amino acids281. The problems arising from optical rotation measurements of chiral purity were reviewed. Important considerations are the nonideal dependence of optical rotation on concentration and the effect of chiral impurities282. 

Hussain A, Faraj J, Aramaki Y, Truelove JE (1985) Hydrolysis of leucine enkephalin in the nasal cavity of the rat- a possible factor in the low bioavailability of nasally administered peptides. Biochem Biophys Res Communl33 923-928. 

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. 

Similarly, chemical hydrolysis of a number of a-amino acyl prodrugs of metronidazole (8.100, R=H see Sect. 8.5.4) was compared to the serum-catalyzed reaction [135][136]. The amino acids used for esterification included alanine, glycine, isoleucine, leucine, lysine, phenylalanine, and valine. Under physiological conditions of pH and temperature, ty2 values for hydrolysis in human serum ranged from 4.5 min for the Phe ester to 96 h for the lie ester. A good linear relationship was established between the log of the rate constant of enzymatic hydrolysis and the log of the rate constant of HO-cata-... 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

Both peak IV (fig. 1) and peak V-2 (fig. 2) resemble cross-links in TLC (Rf = 0 in BUOH/HAC/H2O). IV gave one main peak in SCX-HPLC, but V-2 was separated into several fractions with one major peak (fig. 3b). The yields were relatively low compared with that of HP, namely 1.0, and 1.4 pmoles leucine equivalents for IV and V-2, respectively, versus 10.1 for III (HP). This is not due to instability under acid hydrolysis conditions, since 80% of IV and 100% of V-2 survived acid hydrolysis in a preliminary test. 

Figure 2. Effect of ozone on uptake and incorporation of -leucine into protein by cotton cotyledon leaf discs. Plants were exposed to 0.4 ppm Os for 1 hr, 24 hr prior to experiments. Discs were floated on buffer and incubated in -leucine for up to 4 hr and were then transferred to excess cold leucine to chase the incorporated C-leucine for a subsequent 24 hr period. The data show that ozone-treated tissue incorporated more leucine into protein but do not indicate real differential effects on protein hydrolysis.

By heating a-benzoylamido-yS-isopropylacrylic acid in sealed tubes at 150-170° C., with excess of ammonia, hydrolysis occurred with the formation of leucine, isovalerianic acid and benzoic acid —... 

The results of numerous investigations were published in i860 by Stadeler, who found tyrosine in silk-fibroin, mucin and various other proteins, and who also noted its occurrence in the free state in various organs, generally in conjunction with leucine. Since then, tyrosine has been constantly obtained from proteins by hydrolysis with acids and by the action of trypsin, and has long been regarded as a constituent of the protein molecule. 

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