Thiol groups papain

In cysteine proteinases (CP), activity depends upon a cysteine thiol group. Papain, actinidin, and a few lysosomal cathepsins are the best known members of this family. They hydrolyze peptides and esters in a generally similar fashion to serine proteinases. Two residues are directly involved in the catalytic process, Cys and His, with apparent pKa values of 4.2 and 8.6, respectively. This is the reverse of their normal pKa order, with His being more acidic than Cys. A bell-shaped relation of activity vs. pH for papain is spread out, with maximal activity around pH=6.5 and about half of the activity is retained near 4.5 and 8.5, dropping fast below and above these values. Thus, the active species has a zwitterion state, with a cysteine anion (thiolate) and a histidine cation. CP were discussed in a few reviews (Baker and Drenth, 1987 Fersht, 1985 Polgar and Halasz, 1982)... 

V at pH 7.0 it is not considered very reactive, except e.g. with ascorbate and with NADH bound to lactate dehydrogenase and with the thiol group of cysteine, as shown by the inactivation of papain... 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

Peptide nitriles are reversible inhibitors of cysteine proteases. 1,2 Peptide nitrile reacts with the active site thiol group to form an imidothioate, a dead-end product that does not undergo hydrolysis to an amide.134 This imidothioate derivative has been detected by NMR spectroscopic studies.P 5 The inhibition of papain, a cysteine protease, by a peptide nitrile proved to be reversible in a dialysis experiment. 3 Peptide nitriles are weaker inhibitors of cysteine proteases than the corresponding aldehydes. 61 Most peptide nitriles show poor inhibition toward serine proteases, however those nitriles with proper peptide sequences are potent inhibitors of serine proteases. 7-9 ... 

Cysteine (or thiol) proteases. The /3-thiol group is an even better nucleophile in comparison with the serine side chain thiol proteases operate under alkaline pH values as well examples include papain and bromelain. 

Probably the aspect of primary importance for the catalytic activity of cysteine proteases is the high nucleophilicity of the active-site thiol group. It is now generally accepted that the active form of papain and of cysteine proteases in general consists of a thiolate-imidazolium ion-pair, built from Cys25 and Hisl59. 

Thiol groups in proteins also react with water soluble carbodiimides. For example, papain is modified at cysteine 25. Also, free thiol groups in buckwheat a-glucosidase are protected by reacting them with a carbodiimide. Tyrosine residues are also modified with carbodiimides, and the inactivation of yeast hexokinase is reversed by hydroxylamine. ... 

Their study shows that while 0-methylisourea guanidinates primary amino groups at high pH values (e.g. pH 10.5) more efficiently than it S-methylates thiols, the reverse is true below pH 10. Banks and Shafer (1972) exploited this observation in demonstrating that the thiol group at the active site of papain could he selectively S-methylated at pH 7 without concomitant guanidination of lysyl residues. 

B. S. Baines and K. Brocklehurst. Isolation and characterization of the four major cysteine-proteinase components of the latex of Carica papaya L. Reactivity characteristics towards 2,2 -dipyridyl disulfide of the thiol groups of papain, chymopapains A and B, and papaya peptidase A. J. Protein Chem. 7 119 (1982). 

Structure governing the ionization or pK of cysteine residue will also influence the cysteine oxidation rate [49, 59], As demonstrated using seminal ribonucleases segment 29-34 [60] and a-lactalbumin [61], introduction of an electronegative environment into the structures raises the pK of the thiol groups and reduces the cysteine oxidation rate. In contrast, cysteine residue in papain, which displays a lower pKa due to the presence of a nearby ionizable histidine group, is chemically more reactive than expected [59],... 

Since the NMR resonances of the carbonyl of thioesters are shifted (A 20 to 30 ppm), NMR spectroscopy should allow the direct monitoring of the formation and decay of a thioacyl intermediate. Using = O] N-benzoylimidazole (5 = 168.7 ppm), we were able to observe directly a thioacyl intermediate at 8 = 195.9 ppm in the presence of papain under the cryoenzymological conditions of -t C in 25 percent aqueous dimethyl sulfoxide. Moreover, the thioacyl species is clearly a productive intermediate since the decrease in its signal intensity was accompanied by an increase in the product resonances and by release of free enzyme (half-life, 96 minutes) determined by titration of its thiol group. The line width of the resonance at 195.9 ppm was 25 Hz [5,6]. 

As an example, bromoacetylated flavin analogs were reacted with the thiol group of Cys25 of the enzyme papain to generate artificial flavopapains that catalyzed the oxidation of NADH with higher efficiency than the isolated flavins. 

In an analogous fashion, a flavin-type redox cofactor was attached to the nucleophilic thiol group in the active site of papain. By this means, a hydrolase was transformed into the artificial redox enzyme flavopapain [499-504]. 

The thiol enzyme for which the most detailed mechanistic formulations have been proposed is papain . In this enzyme a cysteine thiol group appears to function in the same manner as the serine hydroxyl of other proteases and esterases. In the hydrolysis of proteins by this plant protease there is an intermediate formation of an acyl thiol, which is subsequently cleaved by water. 

OH attack is shown to occur mainly not at fiee thiol groups, but at tyrosine residues for papain and tryptophan residues for lysozyme and... 

Enzymes that hydrolyze amide and ester bonds may be divided into three classes (1) those requiring a thiol group for activity, such as papain, ficin, and other plant enzymes (2) those inhibited by diisopropylphosphorofluo-ridate (DFP), such as a-chymotrypin, trypsin, subtilisin, cholinesterase, and thrombin (3) those that require a metal ion for activity. This last class includes dipeptidases, and exopeptidases such as carboxypeptidase and leucine aminopeptidase. The metal ion is involved in the stabilization of the tetrahedral intermediate (refer to Section 4.4.1). 

Winnick, Cone, and Greenberg (180) disputed the coenzyme function of activators after demonstrating that no inactivation of ficin occurred after removal of the activators by careful anaerobic dialysis. It is difficult to reconcile these findings with those of Irving et al. Indeed, results with crystalline papain to be presented later give no support to the coenzyme theory and can be interpreted in terms of the reversible oxidation-reduction of thiol groups alone. 

Study of the reaction of the monovalent mercury compounds, p-chloromercuribenzoate (PCMB) and p-chloromercuribenzene-sul-fonate (PCMBS), has provided more information regarding the thiol groups of papain (53,150). In aqueous solution with an excess of PCMB, the combining ratio approaches 1 mole of PCMB per mole of papain as estimated by the spectrophotometric technique of Boyer (34). This PCMB derivative of papain is inactive but is fully reactivated by treatment with cysteine and Versene or with BAL. More important is the fact that the ratio of Hg to protein is propor-... 

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. 

From all evidence, it appears that crystalline papain is homogeneous in the chemical sense. From this standpoint it represents a satisfactory substance for the determination of structure and the correlation of structure and activity. The observation that much of the molecule at the amino-terminal end is not essential for the activity strongly supports the view that enzymes, in general, possess an active site and that intact protein structure is unnecessary for catalytic activity. The presence of an essential thiol group is in accord with the idea that this group is part of the active site and may participate in the catalytic activities of the enzyme through the formation of a thiol ester. 

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