Papain peptide extension

Extensive studies of enzyme-substrate complexes by resonance Raman spectroscopy (RR) have prompted the synthesis of new peptide bond modifications such as thionoesters and dithioesters (Scheme l7)t82-83l within simple model substrates. The resulting acyl-enzyme complexes are especially amenable to RR analysis with cysteine proteases such as papain due to formation of the transient dithioester intermediates. 

Several proteolytic enzymes have a broad substrate specificity, but none are known which will hydrolyze all of the types of peptide bonds found in proteins. The S. griseus proteinase, papain, and the subtilisins extensively hydrolyze most proteins with liberation of free amino acids, but each enzyme also leaves many peptide bonds intact. For total enzymatic... 

From these experiments, we can conclude that the intact papain molecule is not essential for enzyme activity and that the active site of papain is independent of and distant from the amino terminal end of the protein molecule. Just how large the active site must be has still not been demonstrated but the removal of approximately 120 residues of the original 180 suggests that even more extensive degradation may be possible. It has been su ested that the C-terminal portion of papain (Fraction 1) contained the essential thiol grouping because two of the 6 residues of cysteic acid residues reside in this peptide. The observations with LAP-degraded mercuripapain increase the likelihood that Fraction 1 is an int ral part of the active site of papain. 

The development by Bergmann and Zervas (22) of a practical method for the synthesis of a wide variety of peptides and peptide derivatives was followed by extensive studies of the specificity of papain. Hippurylamide was one of the early synthetic substrates found to be rapidly split by papain (23). Later it was found that benzoyl-L-isoglutamine was even more rapidly hydrolyzed (21), and finally Bergmann and Fruton (18) demonstrated that a-benzoyl-L-argininamide (BAA) appeared to be the most sensitive substrate of all. In this sense, papain appeared to resemble trypsin in its specificity since BAA was also the best substrate for this enzyme. However, after investigation of over 65 peptides and peptide derivatives (19-25), it became apparent that the specificity of papain was not nearly so limited as that of trypsin. Bergmann and Fruton (20) pointed out that papain hydrolyzed peptides derived from a wide... 

The use of trypsin rather than papain for the initial proteolytic digestion of the tissue yields dermatan sulfate with a larger peptide moiety, and alkali treatment of such preparations results in destruction of approximately 90% of the serine residues (Stem et al., 1970). The polysaccharide isolated after such treatment is virtually devoid of amino acids, and it may therefore be concluded that serine does indeed participate in the majority of the carbohydrate-protein bonds. The lower degree of serine destruction observed with preparations obtained after more extensive proteolysis is presumably due to the factors indicated above, i.e., that a considerable proportion of the serine residues have free amino or carboxyl groups. 

The order in which the tryptic peptides occur must then be determined by another technique, e.g. according to a scheme of overlapping cleavages with other proteases (pepsin, ch3onotrypsin, papain, and others). The first extensive sequence of amino acids was determined on insulin (Sanger 1954), which is a polypeptide consisting of 51 amino acids. The sequential Analysis of true proteins was still beset with serious obstacles. Finally in 1959, ribonuclease vith 124 amino acid... 

The application of papain in peptide synthesis is well established [23-25]. Papain can be used for fhe preparation of di- and tripepfides in an aqueous medium wifh cosolvent addition (up to 40%) and at high pH to promote synthetic activity. The enzyme is a sulfhydryl protease with no homology to the trypsin or subtilase families of hydrolases. Since the catalytic nucleophile is a cysteine and because thioesters are relatively more prone to aminolysis than oxo-esters, the enzyme could be very attractive for synfhesis. However, unlike the case with the thiol variants of some serine hydrolases, fhe proteolytic activity is still high, and the broad substrate range of proteolysis makes peptide substrate and product hydrolysis more problematic than trypsin or chymotrypsin. Extensive enzyme engineering studies on papain are lacking, probably due to the laborious procedure for isolation of active papain from inclusion bodies formed in E. coli. 

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