Leucine aminopeptidase, protein hydrolysis

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. 

Aminopeptidases are present in many tissues (Table III). Leucine aminopeptidase from intestinal mucosa is very effective in catalyzing the hydrolysis of leucine from the amino terminus of peptides, polypeptides, and proteins. It also hydrolyzes leucine amide and leucine esters (10). The designation leucine aminopeptidase is somewhat of a misnomer because activity is also observed when other amino acids replace leucine. Only the L-isomers of amino acids are substrates, and the presence of a D-amino acid residue or proline in the penultimate position will retard hydrolysis (10). Enzymes having the same specificity as the intestinal aminopeptidase have been identified and/or isolated from kidney, pancreas, muscle, lens, and various bacterial sources (10). The kidney... 

The specificity of this enzyme from swine kidney has been established from detailed studies with synthetic substrates (reviewed by Smith and Hill, 1960). All peptide bonds formed by L-amino acids which are adjacent to a free a-amino group are susceptible to hydrolysis, although the rates of hydrolysis vary over a several thousandfold range. The best substrates are those which contain amino-terminal leucine and the poorest are those which contain the amino nitrogen of proline in peptide linkage, e.g., glycyl-proline (Hill and Schmidt, 1962). The action of leucine aminopeptidase on protein and polypeptide substrates (Hill and Smith, 1958, 1959) agrees with the specificity established with synthetic substrates. 

One of the earliest suggestions that total enzymatic hydrolysis was possible came from the studies of Frankel (1916), who showed that over 90 % of the bonds in several proteins could be broken when proteolysis with pepsin, trypsin, and chymotrypsin was followed by prolonged hydrolysis with the erepsin preparation of Cohnheim (1901). The recognition in later years of several peptidases in intestinal exti acts which will specifically act upon bonds that are not susceptible to the endopoptidases (Bcrg-mann, 1942) probably accounts for these obseiwations. The specific peptidases such as prolidase, iminodipeptidase (prolinase), glycylglycine dipeptidase, tripeptidase, and leucine aminopeptidase, whi( h are present in mucosa, attack many of the bonds that resist the action of endopoptidases. 

Although any of several combinations of proteases can be used, ideally, one or more non-specific endopeptidases should be used first to convert the protein into many small peptides. These small peptides can then be degraded to amino acids by aminopeptidases and prolidase (hydrolyzes X-Pro bonds). Sometimes, carboxypeptidases are also used. Although leucine aminopeptidase has been used as the amino-peptidase (see Hill and Schmidt 1962), it may be preferable to use aminopeptidase M (Rohm and Haas, supplied by Henley and Co. of N.Y.), since this enzyme removes most residues at acceptable rates. Leucine aminopeptidase removes hydrophobic residues most rapidly, whereas some other residues are removed very slowly. Most procedures should probably include the use of prolidase (Miles) since many aminopeptidases do not cleave X-Pro bonds at appreciable rates. If it is found that proline is not released quantitatively by these procedures, the use of citrus leaf carboxypeptidase C (Rohm and Haas) can be tried after the initial endopeptidase hydrolysis and before the addition of aminopeptidase M and prolidase. Carboxypeptidase C (also yeast carboxypeptidase Y - see Hayashi et al. 1973) hydrolyzes proline bonds (as well as all others), but if proline is at or adjacent to the NH2 terminus of a peptide, it would probably not be released. In all procedures a control consisting of the enzymes only should be run in parallel with the hydrolyzed sample, and corrections should be made for any amino acids found by analysis of the control. suhic / /< > , mi... 

Procedure 3 papain, leucine aminopeptidase, prolidase This early procedure for enzymic hydrolysis of proteins was reported by Hill and Schmidt (1962) to be successful for hydrolysis of several proteins. Papain was found to be superior to subtilisin or a combination of trypsin and chymotrypsin for the initial hydrolysis. The method might be improved if aminopeptidase M (discovered after the method was developed) is used in place of the leucine aminopeptidase, but to our knowledge this has not been tested. The problem with diketo-piperazine formation from X-Pro dipeptides in aminopeptidase M hydrolysates of peptides (see above) may make this substitution less desirable than it would seem at first. 

Figure 8 Mechanism of peptide hydrolysis proposed for the leucine aminopeptidase from Aeromonas proteolytica. Protein ligands to the dizinc site are shown only in the first panel (at top left) (reproduced...

Leucine aminopeptidase reacts slowly with Mg++ or Mn++ to form an active enzyme. The rate and extent of activation are functions of metal concentration. The combination with Mn++ is faster than that with Mg++, and results in greater ultimate activity. Nevertheless, the fact that citrate inactivates crude preparations of the enzyme indicates that Mg++, not Mn++, is associated with the enzyme in nature. Citrate inhibits the Mg++-activated, but not the Mn -activated enzyme. The formation of active enzyme is apparently a simple reversible reaction in which one atom of metal combines with one molecule of protein. A dissociation constant could be calculated for the enzyme-Mn combination by measuring the rate of hydrolysis of L-leucinamide as a function of Mn" concentration. The resulting data fit the equation... 

Enzymatic hydrolysis of proteins is an attractive alternative to the hydrolytic procedures and has been used to avoid destruction of tryptophan (174). A mixture of proteolytic enzymes, such as chymo-trypsin, thermolysin, papain, leucine aminopeptidase and pronase has been used (135, 192, 353). Upon completion of the enzymatic hydrolysis of the protein, the hydrolyzate is rendered free of proteolytic enzymes by precipitation and centrifugation and the extract directly analyzed with the amino acid analyzer. The method however may not be generally valid, because of the possibility of artefacts due to self-digestion of the enzymes used. 

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. 

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