Catalyzed hydrolysis, papain

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Figure 1. A simplified representation of the possible process for the papain-catalyzed hydrolysis and aminolysis E, enzyme (papain) S, substrate (protein) ES, Michaelis complex ES, peptidyl enzyme N, nucleophile (amino acid ester) P and P2, products formed from S by hydrolysis ...

Figure 9.28 HPLC profile of papain-catalyzed B AEE hydrolysis product. Conditions column, juBondapak CN eluent, 0.05 M ammonium acetate-methanol (85 15) flow rate, 2.0 mL/min detector, 254 nm at ambient temperature (From Chen et al., 1982.)...

Figure 13. A possible mechanism of action for papain catalyzed hydrolysis...

Carrieri, A., Altomare, C, Barreca, M.L., Contento, A., Carotti, A. and Hansch, C. (1994). Papain Catalyzed Hydrolysis of Aryl Esters A Comparison of the Hansch, Docking and CoMFA Methods. II Farmaco, 49,573-585. 

We have noticed for several systems examined that extrapolations of the Arrhenius plots for intermediate formation to ambient temperatures suggest that the rates of intermediate interconversion will be of the same order of magnitude at the temperatures at which the enzymes normally operate (12). The convergence of Arrhenius plots is shown for the papain-catalyzed hydrolysis of N-carbobenzoxy-L-lysine p-nitroanilide in Figure 3 (28). 

Mammalian liver and muscle frucose bisphosphate aldolases are also very susceptible to limited proteolysis (5,53-55). Cathepsin B, cathepsin L, and papain catalyze the limited proteolysis of rabbit muscle and rat liver aldolases 50,51). In fact, decrease of aldolase activity in liver is observed during starvation 109) and after administration of lysosome-tropic agents 103). Leupeptin caused an increase in osmotic sensitivity of lysosomes and an increase in the activities of free lysosomal proteinases, such as cathepsin A and cathepsin D, and a moderate increase of cathepsin B and L, and resulted in a decrease in aldolase activity. The molecular properties of aldolase isolated from the livers of control rats and leupeptin-treated rats indicated that the decrease of aldolase activity is attributable to hydrolysis of a peptide linkage(s) near the carboxyterminal of the enzyme. However, care is necessary in determining whether proteolytic modification of enzymes... 

Different enzymes have different specificities. Some, such as amylase, are specific for a single substrate, but others operate on a range of substrates. Papain, for instance, a globular protein of 212 amino acids isolated from papaya fruit, catalyzes the hydrolysis of many kinds of peptide bonds. In fact, it s this ability to hydrolyze peptide bonds that makes papain useful as a meat tenderizer and a cleaner for contact lenses. 

This enzyme [EC 3.4.22.25] catalyzes the hydrolysis of peptide bonds with a preference for Gly-Xaa in proteins and small molecule substrates. The enzyme, a member of the peptidase family Cl, is isolated from the papaya plant, Carica papaya. It is not inhibited by chicken cys-tatin, unlike most other homologs of papain. 

All peptidases catalyze the general reaction depicted in Figure C2.2.2, the hydrolysis of a peptide bond. The different peptidases are unique with respect to their specificity that is, their ability to accommodate particular sets of amino acids in the vicinity of a potentially scissile peptide bond. Some peptidases have very broad specificities, such as papain, which has few limi-... 

Proteases such as a-chymotrypsin, papain, and subtilisin are also useful biocatalysts for regio-selective or stereoselective hydrolytic biotransformations. For example, dibenzyl esters of aspartic and glutamic acid can be selectively deprotected at the 1-position by subtilisin-catalyzed hydrolysis (Fig. 6). In addition, a-chymotrypsin is used in the kinetic resolution of a-nitro-a-methyl carboxylates, which results in l-configured enantiomers of the unhydrolyzed esters with high optical purity (>95% e.e.). ... 

The first protease-catalyzed reaction in ILs was the Z-aspartame synthesis (Scheme 10.7) from carbobenzoxy-L-aspartate and L-phenylalanine methyl ester catalyzed by thermolysin in [BMIM] [PF ] [ 14]. Subtilisin is a serine protease responsible for the conversion of A -acyl amino acid ester to the corresponding amino acid derivatives. Zhao et al. [90] have used subtilisin in water with 15% [EtPy][CF3COO] as cosolvent to hydrolytically convert a series of A -acyl amino acid esters often with higher enantioselectivity than with organic cosolvent like acetonitrile (Scheme 10.8, Table 10.2). They specifically achieved l-serine and L-4-chlorophenylalanine with an enantiomeric access (ee) of-90% and -35% product yield which was not possible with acetonitrile as a cosolvent [90]. Another example is hydrolysis of A-unprotected amino acid ester in the presence of a cysteine protease known as papain. Liu et al. 

Crystallographic studies of native cysteine proteinases and enzyme-inhibitor complexes have been used to interpret much of the kinetic data for cysteine proteinase-catalyzed hydrolysis of amide bonds. Analysis of the crystal structures of papain [16], caricain [38], actinidain [56], etc. shows that these structures are closely related. The active site of all these cysteine proteinases contains the Cys-25 sulfhydryl group in close proximity to the His-159 imidazole ring nitrogens, where the latter can abstract the sulfhydryl proton to facilitate attack on the substrate amide carbonyl group [17]. 

Protein Hydrolysates. Instead of ethyl hippurate, a peptic hydrolysate of ovalbumin was used as substrate for the resynthesis reaction (64). This substrate (300 mg) was dissolved in water, adjusted to pH 6.0 with NaOH and to 0.9 ml with additional water. An amino acid ester was added to produce a 22.2mM solution and the mixture preincubated at 37°C for 15 min. Papain (3 mg), dissolved in 0.1M L-cysteine (0.1 ml), was combined with the above-mentioned preincubation mixture and incubation carried out at 37°C. After 2 hr, 0.1N NaOH (10 ml) was added to stop the enzymatic reaction and the resulting solution allowed to stand for 3 hr to hydrolyze completely the remaining amino acid ester as well as the ester group from the peptide product. The free amino acid produced from the base-catalyzed hydrolysis of the amino acid ester was determined with an amino acid analyzer. The amount of the amino acid incorporated was obtained by subtracting the determined value from the initial concentration of amino acid ester. The data obtained with the same L-amino acid esters as used in the model experiment (above) are plotted along the ordinate of Figure 3. An excellent correlation is found between the data from the model experiment and those from this experiment using a protein hydrolysate. In Table III data are shown for the extent of covalent incorporation after 2 hr of various amino acid ethyl esters into the protein hydrolysate. There is a close relationship between... 

Results from CoMFA studies have been compared with those from Hansch analyses [38, 1019 — 1023] and the minimal topological difference (MTD) method [1024]. Examples for the comparison of Hansch equations with CoMFA studies are e.g. the papain hydrolysis of N-(X-benzoyl)glycine pyridyl esters (60) (eqs. 204, 205 Zn = PLS component n of the corresponding field compare chapter 7.1) [1019, 1020], the emulsin-catalyzed hydrolysis of phenyl-P-D-glucosides [1020], the mutagenic activities of substituted (o-phenylenediamine)platinum dichlorides [1020], dihydrofolate reductase (DHFR) inhibition [1020], and some other biological activities [38, 1021—1023]. 

That proteolytic enzymes can indeed catalyze the reversal of proteolysis was shown by Bergmann and Fraenkel-Conrat in 1937 in their study of the synthesis of insoluble amides and anilides catalyzed by papain and bromelin. These studies have been extended recently by other investigators. 113,114 q hg enzymes displayed the same specificity in synthesis as they did in hydrolysis. Moreover, when the free energy of the reaction was lowered by the subseqeunt hydrolysis of the product rather than by precipitation, the synthetic reaction also occurred but the net result of the reaction was a hydrolysis. These experiments serve only as models since in vivo such insoluble products as the anilides are not formed nor would protein synthesis result in net hydrolysis. 

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