Hydrolysis with Specific Endopeptidases

The rate of hydrolysis is affected by the amino acid that follows the basic residue in the sequence. Thus the Arg-Asp bond is cleaved more slowly than most other peptide bonds on the right side of arginine. Similarly, the bond between arginine and cysteic acid (the oxidized form of cysteine) is relatively resistant to tryptic hydrolysis. As it will be shown later such differences can be exploited in sequence studies, but it is possible to create even more substantial differences by appropriate modifications of the peptide molecule. For instance, treatment of a peptide that contains both lysine and arginine residues with maleic acid anhydride or preferably with citraconic acid anhydride yields derivatives in which the lysine residues have no free amino group in their side chain 

Trypsin can also be applied in cysteine containing peptides, because aminoethylation of the sulfhydryl group transforms the cysteine side chain to 

Next to trypsin chymotrypsin is the most preferred proteolytic enzyme in sequencing. Its specificity is less absolute than that of trypsin. Primarily the bonds that follow phenylalanine, tyrosine and tryptophan are cleaved, but measurable hydrolysis takes place next to leucine and methionine residues as well. It is advisable, therefore, to determine in preliminary experiments the conditions (enzyme-substrate ratio, time, temperature) best suited for the formation of a few and well separable fragments. Occasionally also less specific enzymes, such as pepsin, papain or thermolysin find application in structure elucidation. For the hydrolysis of specific bonds new microbial proteases can be isolated. There are known prolidases and also enzymes which hydrolyze solely the bond which follows a pyroglutamyl residue and so on. 

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Endopeptidases. Our expanding understanding of the relationship between structure and functionality of food proteins presents the opportunity for designing functionality into proteins by selective, specific proteolytic modification. Control of reaction and prevention of autolysis offered by immobilization are essential to establish the conditions for a highly selective modification. Hydrolysis at specific positions in the primary structure of proteins could be coupled with resynthesis of peptide bonds by selection of conditions, for example, as in the plastein reaction. By careful choice of enzymes and conditions according to the characteristics of the substrate proteins, it may be possible to design new structures from known food proteins. 

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... 

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments, eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, fi"om the ends of polypeptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from rhe free carboxyl terminal, and aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptides, which are not substrates for exopeptidases, are hydrolyzed in the brush border of intestinal mucosal cells by dipeptidases. 

The mechanism of hydrolysis of cysteine peptidases, in particular cysteine endopeptidases (EC 3.4.22), shows similarities and differences with that of serine peptidases [2] [3a] [55 - 59]. Cysteine peptidases also form a covalent, ac-ylated intermediate, but here the attacking nucleophile is the SH group of a cysteine residue, or, rather, the deprotonated thiolate group. Like in serine hydrolases, the imidazole ring of a histidine residue activates the nucleophile, but there is a major difference, since here proton abstraction does not appear to be concerted with nucleophilic substitution but with formation of the stable thiolate-imidazolium ion pair. Presumably as a result of this specific activation of the nucleophile, a H-bond acceptor group like Glu or Asp as found in serine hydrolases is seldom present to complete a catalytic triad. For this reason, cysteine endopeptidases are considered to possess a catalytic dyad (i.e., Cys-S plus H-His+). The active site also contains an oxyanion hole where the terminal NH2 group of a glutamine residue plays a major role. 

This enzyme [EC 3.4.21.62], a serine endopeptidase that evolved independently of chymotrypsin, contains no cys-teinyl residues. This enzyme catalyzes the hydrolysis of peptide bonds in proteins and has a broad specificity, with a preference for a large uncharged aminoacyl residue in the PI subsite. 

The International Union of Biochemistry and Molecular Biology recommends that the term peptidase be used synonymously with the term peptide hydrolase (IUBMB, 1992). Thus, in this unit the term peptidase is used in reference to any enzyme that catalyzes the hydrolysis of peptide bonds, without distinguishing between exo- and endopeptidase activities. Peptidases may be assayed using native or modified proteins, peptides, or synthetic substrates. In this unit, the focus is on assays based on the hydrolysis of common, commercially available, protein substrates. Thus, the assays are not intended to be selective for a given peptidase they are designed to provide estimates of overall peptidase activity. Other units in this publication focus on synthetic or model substrates, which can be designed for the measurement of specific endo- and/or exopeptidase activities. 

Proteins and peptides are accessible to enzymatic action due to the susceptibility of specific amino acid sequences, and such proteolysis is a naturally occurring metabolic process in vivo. Degradation pathways generally involve hydrolysis of peptide bonds by a variety of exopeptidases and endopeptidases, and the specific proteolytic enzymes associated with non-invasive routes of administration have been identified in some detail. Enzymatic activity varies depending on the delivery route and a qualitative rank ordering is shown in Table 1. Since a significant portion of dietary protein consumed by humans is assimilated by means... 

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