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PubMed1998

An SOD-mimicry mechanism underlies the role of nitroxides in protecting papain from oxidative inactivation.

Nitroxide stable free radicals have previously been found to afford protection in various biological systems against diverse types of oxidative stress, including, ischemia/reperfusion, hyperoxia, mechanical trauma, toxic xenobiotics, ionizing radiation, gastric and colonic irritants or strong oxidants. Dismutation of superoxide has originally been suggested to be one of the mechanisms that underlie the anti-oxidant effect of nitroxides. However, no direct evidence has been found, so far, to support this assumption. In the present study, superoxide and H2O2, generated enzymatically, were used to directly inactivate papain, a sulfhydryl enzyme, in vitro. The rate of papain inactivation served to assess the damage. The reaction mixtures contained a chelate in order to prevent the effect of adventitious redox-active metal ions, pre-empt the Fenton reaction and avoid hydroxyl-induced damage. Catalase or SOD alone partially protected the papain from inactivation. The protective effect of nitroxides resembled that of SOD in several aspects: a) nitroxides provided partial protection; b) the protective effect of nitroxides did not increase with the elevation of their concentration (above 0.5 mM); c) the combined addition of SOD and the nitroxide did not provide greater protection than that demonstrated by nitroxides or SOD separately; d) the effects of catalase with the nitroxide were additive; e) the nitroxide, like SOD itself, did not protect papain from H2O2-induced inactivation; f) the nitroxide was found not to be consumed in the course of the reaction but rather to be recycled. The results indicate that: (a) the main species responsible for the papain inactivation in a system in which the effect of transition metals is pre-empted, are O2-. and H2O2; (b) nitroxides inhibit the oxidative damage by removing superoxide not stoichiometrically, but rather catalytically as SOD-mimics; (c) nitroxides do not afford protection when the oxidative damage is induced directly by H2O2 (and not mediated by redox-active metals).

Antioxidants↗
PubMed2001

Chemically modified papain for applications in detergent formulations.

Papain was modified using succinic anhydride, and the modified papain so obtained was compared with the native papain for its activity and stability in detergents. This study was done using commercial enzyme detergents as references. It was found that modified papain retained activity comparable to the commercial enzyme detergents. Chemically modified papain may prove to be an inexpensive alternative to alkaline proteases that are used in detergents.

Biotechnology↗
PubMed1997

Inhibition of papain with 2-benzyl-3,4-epoxybutanoic acid esters. Mechanistic and stereochemical probe for cysteine protease catalysis.

Papain, a prototypic cysteine protease was inactivated by methyl and benzyl esters of (2S,3S)-2-benzyl-3,4-epoxybutanoic acid. On the other hand, methyl ester of (2S,3R)-2-benzyl-3,4-epoxybutanoic acid was shown to be a competitive inhibitor for the enzyme. It was inferred from the inactivation stereochemistry that in the papain catalytic reaction the nucleophilic attack of the side chain thioalkoxide of Cys-25 on the scissile peptide bond of substrates occurs in the 're' fashion. The papain inactivating potency of (2S,3S)-2-benzyl-3,4-epoxybutanoic acid methyl ester was enhanced over three-fold in a pH 8.0 solution compared with in the neutral solution. This together with our previous observation with alpha-chymotrypsin and the recent theoretical treatment of the enzymic reaction of papain, suggest that in the inactivation of papain by oxirane containing inhibitors, the oxirane does not need to be activated by prior protonation as thought previously. The oxirane ring is sufficiently labile that the unprotonated oxirane moiety can undergo an electrophilic reaction with the Cys-25 thiolate.

Binding Sites↗
PubMed1991

Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain.

The existence of an oxyanion hole in cysteine proteases able to stabilize a transition-state complex in a manner analogous to that found with serine proteases has been the object of controversy for many years. In papain, the side chain of Gln19 forms one of the hydrogen-bond donors in the putative oxyanion hole, and its contribution to transition-state stabilization has been evaluated by site-directed mutagenesis. Mutation of Gln19 to Ala caused a decrease in kcat/KM for hydrolysis of CBZ-Phe-Arg-MCA, which is 7700 M-1 s-1 in the mutant enzyme as compared to 464,000 M-1 s-1 in wild-type papain. With a Gln19Ser variant, the activity is even lower, with a kcat/KM value of 760 M-1 s-1. The 60- and 600-fold decreases in kcat/KM correspond to changes in free energy of catalysis of 2.4 and 3.8 kcal/mol for Gln19Ala and Gln19Ser, respectively. In both cases, the decrease in activity is in large part attributable to a decrease in kcat, while KM values are only slightly affected. These results indicate that the oxyanion hole is operational in the papain-catalyzed hydrolysis of CBZ-Phe-Arg-MCA and constitute the first direct evidence of a mechanistic requirement for oxyanion stabilization in the transition state of reactions catalyzed by cysteine proteases. The equilibrium constants Ki for inhibition of the papain mutants by the aldehyde Ac-Phe-Gly-CHO have also been determined. Contrary to the results with the substrate, mutation at position 19 of papain has a very small effect on binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Conformational variability in an enzyme's active site: resonance Raman evidence for different acyl group conformations in N-acylglycine and N-acylalanine dithioacyl papains.

It is demonstrated that the vibrational modes associated with the catalytically labile region of N-acylalanine dithioacyl papains undergo a major reorganization compared to the normal modes of corresponding model compounds. Thus, the resonance Raman (RR) spectrum of, e.g., N-benzoylalanine dithioacyl papain and its response to isotopic labeling cannot be understood completely on the basis of the RR spectrum of N-benzoylalanine ethyl dithio ester in one of its known conformational states [detailed in Lee, H., Angus, R. H., Storer, A. C., Varughese, K. I., & Carey, P. R. (1988) Biochemistry (preceding paper in this issue)]. This situation contrasts sharply to that for N-acylglycine dithioacyl papains whose RR spectra closely resemble those of the corresponding N-acylglycine ethyl dithio esters in a conformational state known as conformer B. For the N-acylalanine intermediates two possible causes are put forward to explain the rearrangement of the normal modes. First, the acyl groups based on alanine may bind in papain's active site in a conformation whose torsional angles near the -C(=S)S-group differ markedly from those of characterized model compounds. The second, and presently favored, explanation is that the N-acylalanine moiety is binding in the active site in an A- or C5-like conformation and that, in addition, there is significant vibrational coupling between some of the normal modes of the bound substrate and the normal modes associated with parts of the enzyme in contact with the substrate. The finding that deacylation for N-acylglycine or N-acylalanine dithioacyl papains must proceed from structures which are different is an indication that the mechanism of deacylation may not have strict stereochemical requirements.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1981

Determination of a low pK for histidine-159 in the S-methylthio derivative of papain by proton nuclear magnetic resonance spectroscopy.

Proton NMR spectroscopy was used to study the ionization behavior of His-159 in a derivative of papain (papain-S-SCH3). In this catalytically inactive derivative of papain, the active-site thiol group of Cys-25 is S-methyl-thiolated so that it cannot form a thiolate anion. The pH dependence of the chemical shift of the C epsilon 1 H resonance of His-159 indicated a pK of 3.45 +/- 0.07 at 45 degrees C in 2H2O with no added ions other than those required for titration. In acetate buffers at an ionic strength of 0.05, the pK increased to 3.87 +/- 0.12. Conversion of papain-S-SCH3 to active papain at pH* 4.17 (at 45 degrees C and an ionic strength of 0.05) caused the position of the C epsilon 1 H resonance to change from a position indicative of partial protonation of His-159 to a position indicative of full protonation, consistent with the existence of an imidazolium-thiolate ion-pair interaction between His-159 and Cys-25 in the active enzyme.

Histidine↗
PubMed1978

Effects of substituents on the rates of deacylation of substituted benzoyl papains. Role of a carboxylate residue in the catalytic mechanism.

The effect of ring substituents on the rates of deacylation of 8 meta- and para-substituted benzoyl papains was evaluated. The rate constants were found to depend upon a single ionizing group of pKa = 4.2--4.3, and to decrease by a factor of approximately 2.2 when measured in 94% D2O/H2O. The rates of deacylation are increased greatly by electron-withdrawing groups on the benzene ring. The Hammett rho value is 2.74 +/- 0.32. A plot of the rate constants for deacylation of the benzoyl papains against the corresponding constants for substituted benzoyl chymotrypsins generates a straight line of slope 1.0. This result suggests a very similar distribution of charge on the benzoyl moiety in the transition state for the two enzymes, which is interpreted in terms of the net charge of the transition state for the deacylation of nonspecific acyl papains being equal to--1 with the general base catalyzed assistance to the attack of water on the acyl enzyme being provided by the negatively charged Asp-158 rather than by the neutral Asn-175-His-159 hydrogen bond network. This result together with a survey of literature data suggests that the role of Asp-158 in papain catalysis has been underestimated. The evidence advanced to date in support of the proposition that an imidazolium-159-cysteine-25 thiolate ion pair exists in native papain is evaluated and considered to be insufficient to decide the issue.

Benzoates↗
PubMed1976

The chemical modification of papain with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The reaction of the water-soluble carbodimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), with active papain in the presence of the nucleophile ethyl glycinate results in an irreversible inactivation of the enzyme. This inactivation is accompanied by the derivatization of the catalytically essential thiol group of the enzyme (Cys-25) and by the modification of 6 out of 14 of papain's carboxyl groups and up to 9 out of 19 of the enyzme's tyrosyl residues. No apparent irreversible modification of histidine residues is observed. Mercuripapain is also irreversibly inactivated by EDC/ethyl glycinate, again with the concomitant modification of 6 carboxyl groups, up to 10 tyrosyl residues, and no histidine residues; but in this case there is no thiol derivatization. Treatment of either modified native papain or modified mercuripapain with hydroxylamine results in the complete regeneration of free tyrosyl residues but does not restore any activity. The competitive inhibitor benzamidoacetonitrile substantially protects native papain against inactivation and against the derivatization of the essential thiol group as well as 2 of the 6 otherwise accessible carboxyl groups. The inhibitor has no effect upon tyrosyl modification. These findings are discussed in the context of a possible catalytic role for a carboxyl group in the active site of papain.

Amino Acids↗
PubMed2002

The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells.

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.

Amino Acid Sequence↗
PubMed2000

Polyclonal-antibody-mediated insolubilization and stabilization of papain.

Antisera raised in rabbits against native iodoacetamide- and iodoacetic acid-modified papain contained precipitating and non-inhibitory antibodies. Papain could be insolubilized as enzyme-antibody adducts with the gamma-globulin fraction derived from the antiserum. Two insolubilized papain preparations, designated A and B, with low and high antibody/enzyme ratios, respectively, exhibited high enzyme activity and markedly enhanced thermal and denaturant stabilities. However, papain antibody adduct B was superior in activity and stability over preparation A. The usefulness of immobilized papain in biopharmaceutical and other industries is also discussed.

Animals↗
PubMed1968

Evidence for histidine in the active site of papain.

Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2-(14)C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.

Acetone↗
PubMed1993

Ionization characteristics of the Cys-25/His-159 interactive system and of the modulatory group of papain: resolution of ambiguity by electronic perturbation of the quasi-2-mercaptopyridine leaving group in a new pyrimidyl disulphide reactivity probe.

1. A new thiol-specific reactivity probe 4,4'-dipyrimidyl disulphide [compound (VII), m.p. 110 degrees C, pKa of its monohydronated form 0.91] was synthesized and used to resolve the ambiguity of interpretation of the behaviour of papain (EC 3.4.22.2) in alkaline media known to depend to varying extents on two ionizations with pKa values approx. 8.0-8.5 and > or = 9.5 respectively. 2. A new extensive pH-second-order rate constant (k) data set for the reaction of papain with 2-(acetamido)-ethyl 2'-pyridyl disulphide (IV) demonstrated the existence of a striking rate maximum at pH approx. 4, the independence of k around pH 8 and the increase in k with increase in pH across a pKa value of 10.0, behaviour similar to that of other 2-pyridyl disulphides (R-S-S-2-Py) that lack key substrate-like binding sites in R. 3. Although the simplest interpretation of the pKa value of 10.0 assigns it to the formation of (Cys-25)-S-/(His-159)-Im from the ion-pair state of the papain catalytic site, another interpretation may be conceived in which this pKa value is assigned to another group remote from the catalytic site, the state of ionization of which modulates catalytic-site behaviour. This alternative assignment is shown to require compensating effects in the pH region around 8 such that the formation of (Cys-25)-S-/(His-159)-Im across pKa 8.0-8.5 is without net kinetic effect in the reactions of simple 2-pyridyl disulphides such as compound (IV) and 2,2'-dipyridyl disulphide (II). 4. The lower basicity of compound (VII) relative to that of compound (II) (pKa 2.45) was predicted to diminish or abolish the compensation postulated as a possibility in reactions of 2-pyridyl disulphides because of the decreased effectiveness of reaction via a (His-159)-Im+H-assisted transition state. The characteristics of the pH-dependence of the reaction of papain with compound (VII) which are quite different from those for its reaction with compound (II) support both this prediction and the alternative assignment with a value of 8.3 for the pKa of the formation of (Cys-25)-S-/(His-159)-Im. 5. Evidence that the behaviour of papain towards both substrates and some substrate-derived time-dependent inhibitors is determined not only by the loss of the (Cys-25)-S-/(His-159)-Im+H ion-pair state by dehydronation with pKa 8.3 but also by another ionization of pKa approx. 10.0 is briefly discussed.

Binding Sites↗
PubMedDate not supplied

Heterogeneity of anti-U demonstrable by the use of papain-treated red cells.

When red cells (RBCs) are treated with papain, one form of the U antigen, which we have named UPS (U papain-sensitive), is almost completely removed or denatured. A second form, UPR (U papain-resistant), remains unaltered on the treated RBCs. Tests on 42 examples of anti-U showed that two contained only anti-UPS, 19 contained only -UPR, and 21 contained separable -UPS and -UPR. In those sera containing both antibodies, anti-UPR was always the stronger of the two. These findings suggest 1) that UPS is located on the Ss sialoglycoprotein (glycophorin B) at a position distal to a papain-sensitive site or that the cleavage point is within the portion of the SGP that comprises UPS, and 2) that UPR is located between the papain-sensitive site and the RBC membrane. The UPS determinant was not denatured by neuraminidase, L-cysteine, trypsin, ficin, or alpha-chymotrypsin, and it was only partially denatured by pronase. The finding that RBCs treated with para-chloromercuribenzoic acid or para-chloromercuriphenyl sulfonic acid did not react with anti-UPR but did continue to react with anti-UPS suggests that the in situ configuration of UPR, but not UPS, is dependent on the presence of one or more disulfide bonds. RBCs of the S-s-U+(weak) phenotype were shown to carry markedly reduced amounts of both UPS and UPR.

Adsorption↗
PubMed1990

Comparative study on specificities of rat cathepsin L and papain: amino acid differences at substrate-binding sites are involved in their specificities.

Sixty-nine rat cathepsin L-susceptible peptide bonds were analyzed employing various peptide substrates. The proteolytic specificities of rat cathepsin L and papain were compared and the results are discussed in relation to differences in amino acid residues around their binding sites. The specificity of cathepsin L, which is characterized by a remarkable preference for hydrophobic amino acids at the P2 site of the scissile peptide bonds, was analogous to that of papain as a whole. This analogous specificity suggests that the binding sites of the two proteases are analogous, as expected from their homologous amino acid sequences. However, there is a slight difference in the preference for S3 site between them. That is, cathepsin L showed a greater preference for bulky and hydrophobic amino acids at the S3 site than did papain. Based on the computer-graphically deduced structure of the binding sites of cathepsin L, the preferences for hydrophobic amino acids at the S2 site and for bulky and hydrophobic amino acids at the S3 site of the protease are supposed to be related to the compensating amino acid substitutions at the S2 site (V133A and V157L) and the reduction in size at the S3 site (Y61Q and Y67L), respectively. The discussion of the effect of the amino acid substitutions on the proteolytic activities of cathepsin L and papain in this paper provides a basis for more advanced studies of the relationship between structure and function of proteases belonging to the papain superfamily by means of protein engineering.

Amino Acid Sequence↗
PubMed1978

Modification of papain with tetranitromethane.

Papain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spectra of polymerized papains showed a small change in ellipticity when compared with that of unmodified papain. Esterolytic activity of the modified enzyme toward benzoyl-L-arginine ethyl ester (BAEE) was almost fully retained, at least up to the formation of hexamer, with an unchanged Km value. Spectrophotometric and amino acid analyses indicated that two or three tyrosine residues are involved in intermolecular crosslinks depending on the amount of TNM used. The tyrosine residues nitrated were identified as those at positions 61, 116, 103, and 4, the extent of nitration decreasing in this order. When activated papain was treated with increasing molar ratios of TNM, an essential sulfhydryl function was first oxidized and, at a 2-fold molar excess of the reagent, restoration of activity was no longer observed even after addition of dithiothreitol (DTT). The evidence obtained in the present study eliminates the possibility of inactivation due to nitration of a tryptophan residue, which had been suggested previously.

Amino Acid Sequence↗
PubMed1991

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain.

A mutant of papain, where an inter-domain hydrogen bond between the side chain hydroxyl group of a serine residue at position 176 and the side chain carbonyl oxygen of a glutamine residue at position 19 has been removed by site-directed mutagenesis, has been produced and characterized kinetically. The mutation of Ser176 to an alanine has only a small effect on the kinetic parameters, the kcat/Km for hydrolysis of CBZ-Phe-Arg-MCA by the Ser176Ala enzyme being of 8.1 x 10(4) /M/s compared with 1.2 x 10(5) /M/s for papain. Serine 176 is therefore not essential for the catalytic functioning of papain, even though this residue is conserved in all cysteine proteases sequenced. The pH-activity profiles were shown to be narrower in the mutant enzyme by up to 1 pH unit at high ionic strength. This result is interpreted to indicate that replacing Ser176 by an alanine destabilizes the thiolate-imidazolium form of the catalytic site Cys25-His159 residues of papain. Possible explanations for that effect are given and the role of a serine residue at position 176 in papain is discussed.

Base Sequence↗
PubMed1976

Effect of the immediate environment on the reactivity of the essential -SH group of papain.

The effect of the microenvironment on the reactivity of the essential -- SH group of papain was studied by alkylation with methyl iodide and with the more polar iodoacetamide. Rate and activation parameters for these reactions were determined with two forms of the -- SH group: the free mercaptide ion at pH 10.0, and the mercaptide-imidazolium ion-pair at pH 5.5. The ion-pair of papain reacts with methyl iodide at a rate 1470 times less than that of thiolsubtilisin. This surprising difference between the reactivities of the two enzymes suggests that in contrast to thiolsubtilisin, where a non-polar environment enhances the rate, in the case of papain a more polar environment somewhat inhibits the reaction with the non-polar methyl iodide. The positive activation entropy for the papain reaction may indicate an 'ordered' structure of bound water around the sulfur atom. The high rate and the low activation entropy (organized transition state) of the reaction of papain with iodoacetamide can be explained in terms of hydrogen-bond formation between the enzyme and the amide group of the alkylating agent.

Alkylation↗
PubMed1977

Inhibition of papain by N-acyl-aminoacetaldehydes and N-acyl-aminopropanones. Evidence for hemithioacetal formation by a cross-saturation technique in nuclear-magnetic resonance spectroscopy.

N-Acyl-aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitor properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N-acetyl-aminoacetaldehyde-papain complex. Using a cross-saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active-site thiol group. Hemithioacetal formation has also been detected between N-benzoyl-aminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain-catalysed hydrolyses.

Acetaldehyde↗