Specificity of papain

Figure 5. Specificity of papain in relation to its active site (67)...

Specificity of Papain, for Hydrolysis of Peptide Bonds in Proteins and Polypeptides ... 

It can be concluded that the wide specificity of papain observed in studies with synthetic substrates is in agreement with the results of studies on small polypeptides. On the other hand, it will be difficult to predict which types of bonds will be hydrolyzed in a peptide of known composition, inasmuch as several structural features around a bond formed by an amino acid with a structurally favorable side chain must determine the rate of hydrolysis. 

The concept of enzyme subsites for binding of the several amino acid residues on either side of the scissile bond to the enzyme was introduced first by Schechter and Berger (27, 28) from their studies on the substrate specificity of papain. This concept of subsites will have special relevance to the specificity of amino acid incorporation during the resynthesis reaction to be discussed later. 

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 fact that the preparations of papain used in these investigations might contain more than one proteolytic em me was never overlooked by any of the investigators, but it prevented a conclusive description of the specificity of papain. Nevertheless, the results were in agreement with earlier observations with protein substrates. 

Fox and his associates (54-58) have studied the effect of a number of factors on anilide synthesis by papain. They report that the optimal pH for anilide synthesis varies with the A-acyl amino acid used. The influence of the amino acid side chain influences the rate of anilide synthesis much as would be expected from the hydrolytic specificity of papain. 

Johnston,. Mycek, and Fruton (95) extended these observations and found that transamidation with papain proceeded more rapidly at pH 8 than at pH 5. Furthermore, hydroxylamine was found to be an effective replacement agent, yielding hydroxamic acids. The specificity of papain toward the substrate in these reactions was similar to that for the hydrol rtic reaction. In a later study (97) it was demonstrated that amino acid amides or peptides could serve as replacing agents, e.g., papain catalyzed the following synthesis ... 

Formation of a thiol ester is consistent with the spatial and chemical specificity of papain which is toward the amino acid residue bearing the carbonyl group of the sensitive bond. The moiety which is displaced from, or transferred to, the carbon of this carbonyl group may be any of the compounds mentioned above, e.g., a peptide, an amino acid, ammonia, aniline, hydrazine, hydroxylamine, an alcohol,... 

There has been no attempt to explain all the features of papain specificity nor to indicate the number of steps from I to IV in the scheme of Figure 28. Indeed, it is likely that other groups on the substrate also interact with the enzjrme. Postulation of a three-point interaction may be necessary to explain the optical specificity of papain. The scheme of Figure 28 may be regarded as a minimal working hypothesis. 

The broad specificity of papain, which is in contrast to the narrower specificity of the crystalline proteinases of animal origin, no longer appears to be the puzzle it once was. Other crystalline proteinases, such as subtilisin (167) and pepsin (138,141) also appear to possess a broad spectrum of action. Moreover, several exopep-... 

Next we evaluate the PDLD + EVB surface for the enzymatic reaction using eq. (5.17). The resulting surface is shown in Fig. 5.6. As seen from the ligure, the protein can reduce Aby stabilizing the ionic state more than water. In fact, in the specific case of papain the protein inverts the stabilization of the covalent and ionic states relative to their order in solution. 

In another approach the specificity of an existing enzyme has been changed. Levine and Kaiser (57) have transformed a protease, papain, into a redox enzyme by alkylation of the active site thiol with (27), a derivative of xanthine. 

This enzyme [EC 3.4.22.6], also known as papaya proteinase II, is a member of the peptidase family Cl. It is the major endopeptidase of papaya (Carica papaya) latex. It has a specificity similar to that of papain. In addition, there are a number of chromatographic forms of the enzyme. 

Jacquct et al. described the proteolytic specificities of chymopapain and papaya proteinase Q [36]. The polypeptide chain of caricain contains 216 amino acid residues (Mr 23283). The molecule shares 148 identical amino acid residues (68,5ft) with papain. 141 with chymopapain (65,2ft), and 175 with glycyl endopeptidase (81.0ft). The Ms,lllOnin is 1U [20]. The three-dimensional structure of caricain has been determined by x-ray diffraction analysis [37,38]-... 

Hie Si sub site has no binding pocket as such and has a much less clearly defined specificity, expect for glycyl endopeptidase. Low molecular weight substrates indicate some preference for Lys or Arg (over Ala) at P] of papain [3], Schechter and Beiger [68] showed that Leu and Phe bind equally well at Sit indicating that the interaction is probably mainly hydrophobic. It is striking, however, that Aftl is not accepted in Si [49]. 

K. I. Angeuoes and A. L, rink, Mechanism of action of papain with a specific anilide substrate. Biochemistry J 235J (1979),... 

Stem bromelain has a broad substrate specificity, dose to the specificity of ficin (EC 3.4.22.3), end hydrolyzes a great variety of synthetic and natural substrates. It preferentially cleaves peptide bonds when a hydrophobic group is in the rz position. (According to Scheduler and Betger [32], the amino add residues in a substrate undergoing cleavage are designated as Pi, P2, P3, etc. in the N-tennmal direction and Pi, Y P3r, etc. in the C-temunal direction from the cleaved bond.) Predominantly Gly-Fhe, Phe-Ser, Tyr-Ile, and "iyr-Val bands are cleaved [33,34]. The specificity of bromelain is different from that of papain and ficin in that it hydrolyzes most effectively derivatives of Lys, Ala, T r, Gly, and Asn [35]. 

The specific activity of papain is determined using the synthetic peptide substrate N-benzoyl-L-aigmine ethyl ester. Hydrolysis is carried out at pH 7.0 and 25 X and die consumorion of base, necessary to neutralize the lihemiwl acid, is recorded as a function of time. 

I. SCHECHTEE, A. Bergee On the active site of proteases. 3. Mapping the active site of papain specific peptide inhibitors... 

Chaiken, I.M., Smith, E.L. (1969). Reaction of a specific tyrosine residue of papain with diisopropylfluorophosphate. J. Biol. Chem. 244 4247-50. 

It has already been remarked that most enzymes with elastolytic activity have proved to be proteinases with a wide peptide-bond specificity. Thus papain, bromelin, and ficin have a similarly broad hydrolytic action and are all active elastolytic enzymes. Sanger et al. (1955) found that the oxidized A chain of insulin was hydrolyzed in a variety of positions by either crude papain or activated mercuripapain. There were five major... 

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