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PubMed1995

Structural and functional roles of asparagine 175 in the cysteine protease papain.

The role of the asparagine residue in the Cys-His-Asn "catalytic triad" of cysteine proteases has been investigated by replacing Asn175 in papain by alanine and glutamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters determined against the substrate carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)- L-arginine. At the optimal pH of 6.5, the specificity constant (k(cat)/KM)obs was reduced by factors of 3.4 and 150 for the Asn175-->Gln and Asn175-->Ala mutants, respectively. Most of this effect was the result of a decrease in k(cat), as neither mutation significantly affected KM. Substrate hydrolysis by these mutants is still much faster than the non-catalytic rate, and therefore Asn175 cannot be considered as an essential catalytic residue in the cysteine protease papain. Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn175 side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alteration of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the asparagine residue to the structural integrity of papain. The strict conservation of Asn175 in cysteine proteases might therefore result from a combination of functional and structural constraints.

Amino Acid Sequence↗
PubMed1995

The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases.

The replicase ORF1a polyprotein of equine arteritis virus, a positive-stranded RNA virus, is proteolytically processed into (at least) six nonstructural proteins (Nsp). A papain-like Cys protease in Nsp1 and a chymotrypsin-like Ser protease in Nsp4 are involved in this process. In this paper we demonstrate that the Nsp2/3 junction is not cleaved by either of these previously described proteases. Comparative sequence analysis suggested that an additional Cys protease resided in the N-terminal Nsp2 domain. For equine arteritis virus, this domain was shown to induce Nsp2/3 cleavage in a trans-cleavage assay. Processing was abolished when the putative active site residues, Cys-270 and His-332, were replaced. Other Nsp2 domains and three other conserved Cys residues were also shown to be essential. The Nsp2 Cys protease displays sequence similarity with viral papain-like proteases. However, the presumed catalytic Cys-270 is followed by a conserved Gly rather than the characteristic Trp. Replacement of Gly-271 by Trp abolished the Nsp2/3 cleavage. Conservation of a Cys-Gly dipeptide is a hallmark of viral chymotrypsin-like Cys proteases. Thus, the arterivirus Nsp2 protease is an unusual Cys protease with amino acid sequence similarities to both papain-like and chymotrypsin-like proteases.

Amino Acid Sequence↗
PubMed2002

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.

Amino Acid Sequence↗
PubMed1978

Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions.

Losses in enzyme activity and sulphydryl content have been studied in aerated papain solutions containing formate, superoxide dismutase and dithiothreitol. Both formate and dithiothreitol converted .OH to .O2-, whereas superoxide dismutase completely suppressed the inactivation by .O2-. Using results from all three systems, the fraction of .O2- reactions with papain that caused inactivation of the enzyme was 0.33 +/- 0.07. The results also showed that the fraction of .OH reactions, which cause inactivation of papain, is significantly higher in aerated than in oxygen-free solutions.

Cobalt Radioisotopes↗
PubMed1960

Immunological studies of human gamma-globulin. Relation of the precipitin lines of whole gamma-globulin to those of the fragments produced by papain.

Two major antigenic fragments were obtained from various purified gamma-globulin preparations after papain treatment. One, the F component, had a mobility faster than the original gamma-globulin and the second, the S component, had a slower mobility. Similar F and S components were also obtained with certain homogeneous myeloma proteins which were closely related to gamma-globulin immunologically. Additional minor antigenic components were detected with certain antisera. The technique of immunoelectrophoresis was particularly useful for bringing out the different antigenic constituents obtained after papain treatment. The F and S components as well as a midfraction were isolated by chromatography on DEAE-cellulose. These were immunologically homogeneous and could be utilized to absorb F and S antibodies from various antisera. The relative amount of F and S antibodies varied in different antisera from individual rabbits immunized with whole gamma-globulin. Whole gamma-globulin was separated by zone electrophoresis into a fast migrating and a more slowly migrating fraction. Each of these gave rise to F and S components after splitting with papain. The F components of the two gamma-globulins were similar in mobility, while the S components showed marked mobility differences although antigenically they were very similar. The mobility differences of the parent gamma-globulin appeared to be primarily related to the differences in the S component. Certain antisera against pathological gamma-globulins were found to give double lines with a wide variety of gamma-globulin preparations in agar diffusion. These were shown to be related to the F and S antigenic determinants of gamma-glubulin. This relationship was evident by a number of procedures utilizing both Ouchterlony plate techniques and immunoelectrophoresis. The question of whether these findings indicate heterogeneity of gamma-globulin in relation to the F and S antigenic components, or whether different antigenic groups on one molecule can give rise to separate lines in certain instances, is discussed.

Antibodies↗
PubMed1968

Papain digestion fragments of human IgM globulins.

Papain digestion of two Waldenström IgM globulins produced a high amount of small peptides and resulted in the formation of two end products, the Fabmicro and Fcmicro fragments. The Fcmicro fragment is characterized by a fast electrophoretic mobility, a high content in carbohydrate, and a high molecular weight. It was demonstrated that this fragment is made of heavy chain pieces belonging to several disulfide-linked monomeric subunits, presumably representing the carboxy-terminal end of the micro-chains. Fc fragments from the two macroglobulins could not be distinguished immunologically. An appreciable proportion of IgM molecules apparently underwent degradation without the formation of a stable Fc fragment. An Fc-like fragment, analogous to the reduced Fc fragment, was obtained at early stages of papain digestion of the IgM subunits. The Fabmicro fragment, with slow and individually distinct electrophoretic mobility, bears many physicochemical and immunological similarities to the Fabgamma fragment. It consists of one light chain and one Fd piece, both of which were isolated. The interaction of these two constituents was demonstrated by gel diffusion studies. Fab fragments of both IgM globulins were resolved into two subpopulations with different electric charges. In addition to these fragments, intermediary split products were observed at early stages of the degradation process, together with a high yield of small peptides mainly derived from the papain-sensitive region of the heavy chains. Immunologic data strongly suggested that this segment of micro-chains is situated between the Fd piece and the portion included in the Fc fragment. Several experiments indicated the importance of conformational antigenic specificity in both Fab and Fc regions of the IgM globulins.

Carbohydrates↗
PubMed1991

Rat cathepsin H-catalyzed transacylation: comparisons of the mechanism and the specificity with papain-superfamily proteases.

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.

Amino Acid Sequence↗
PubMed1980

Coupling between Cbz-Arg-OH and Leu-X catalyzed by trypsin and papain.

Trypsin [EC 3.4.21.4] and papain [EC 3.4.22.2] catalyzed the coupling between Cbz-Arg-OH and Leu-X (X=-NH2, -OEt, -OBut, or -NH) in solution. The optimum pH for synthesis by trypsin and by papain was 6.5-7.0 and 5.5, respectively. Papain possessed reactivity several times higher than that of trypsin. To shift the reaction toward synthesis, the concentration of amine components must be at least several times higher than that of the carboxyl component (Cbz-Arg-OH). The effect of the concentration of Leu-X depended upon the nature of X; higher concentrations were required in the following order: -NH2 > -OEt, -OBut > -NH. High concentrations of organic cosolvents were also significant for promoting the synthesis.

Arginine↗
PubMed1992

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.

Amino Acid Sequence↗
PubMed1994

The double catalytic triad, Cys25-His159-Asp158 and Cys25-His159-Asn175, in papain catalysis: role of Asp158 and Asn175.

The 1.65 A X-ray structure of papain, which exhibits a Cys25-His159-Asn175 triad, does not correspond to the catalytically active ion pair state since Cys25 is oxidized to cysteic acid and His159 is predominantly neutral. Thus, stochastic boundary molecular dynamics simulations starting from the 1.65 A X-ray structure of papain have been performed for Cys25 and His159 in the SH-ImH+, SH-Im, S(-)-ImH+ and S(-)-Im states and for Asp158 mutated to Asn, Glu and Gly in the ion pair state. By comparing the resulting averaged structures and analyzing the trajectories of certain interatomic distances, important differences in the active-site geometry of papain have been found. In particular, the initial Cys25(S-)-His159(ImH+)-Asn175(C = O) triad found in the X-ray structure is retained in all the structures except the wild type and Asp158-->Asn ion pair states where there is a conformational transition to form the triad, Cys25(S-)-His159(ImH+)-Asp158(COO-). Both triads, Cys25(S-)-His159(ImH+)-Asp158(COO-) and Cys25(S-)-His159(ImH+)-Asn175(C = O) are postulated to participate in catalysis and their roles are discussed. Thus, catalysis does not take place from a single steric position but a two-state mechanism.

Amino Acid Sequence↗
PubMed1994

Modulation of the enzymatic activity of papain by interdomain residues remote from the active site.

The two main catalytic residues Cys25 and His159 of the monomeric cysteine protease papain are located on different walls of a cleft formed by two domains. This topology suggests a possible relationship between relative domain organization and catalytic mechanism. The effect on enzymatic parameters of structural modifications at various locations of the two-domain interface of papain was examined by individual or double replacements by Ala of pairs of interacting residues. Most modifications had no effect on enzyme activity. However, the enzyme's substrate turnover (kcat) decreased following simultaneous alteration of the two most conserved residues, forming an apolar contact located 15 A away from the active site. The pH activity profile of the double mutant was unchanged, indicating a conserved ionization state of the active site thiolate-imidazolium ion pair. This state is strongly dependent on the distance separating the two residues, thus suggesting that the active site geometry has not been significantly altered. Efficient enzymatic activity in papain requires more than a correct active site geometry and is influenced by domain packing properties in a region remote from the active site.

Base Sequence↗
PubMed2006

High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei.

Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 A, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Comparisons reveal that these peptides bind in the active site in a manner similar to that of the human cysteine protease inhibitor stefin B when it is complexed to papain. The assignment of the fragment sequences is consistent with the specificity of the protease.

Animals↗
PubMed2000

The N-terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two-step binding by rapid-kinetic studies of cystatin A labeled at the N-terminus with a fluorescent reporter group.

The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS- and AEDANS-labeled cystatin A to papain resulted in approximately 4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.

Animals↗
PubMed1987

Sensitive two-stage papain technique without cell washing.

A two-stage papain technique is described in which cell washing after papain treatment is replaced by the addition of a specific papain inhibitor. This technique permits optimal enzyme treatment of red cells while digestion of immunoglobulin following the addition of serum is avoided. The technique therefore combines the design and consequent sensitivity advantage of two-stage tests with the convenience of one-stage tests, rendering it suitable for use in compatibility testing.

Antipain↗
PubMed1987

The pH optima for papain and bromelain treatment of red cells.

The action of papain and bromelain, prepared over a pH range from 4.6 to 8.6, was evaluated for the ability to render red cells agglutinable by five incomplete antibodies of differing blood group specificities using a two-stage technique. The optimal pH for treatment of red cells by activated papain or bromelain was between 5.4 and 5.8. Above this pH range, a fall in serological sensitivity was apparent which was much more pronounced with papain than with bromelain. The optimal pH for enzyme treatment of red cells can be achieved in two-stage techniques, but not in one-stage techniques due to the buffering effect of serum proteins.

Agglutination↗
PubMed1976

Proton equilibria in the binding of Zn2+ and of methylmercuric iodide to papain.

The proton liberation on the binding of zinc chloride and methylmercuric iodide to the (essential) thiol group of papain has been examined as a function of pH. This was carried out by (a) direct titration of the protons on the addition of the metal compound to active papain and (b) measurement of the extent of inhibition of enzyme activity by the metal compound as a function of pH. It was found that in the neutral pH range the thiol group or the neighbouring imidazole group in the free enzyme carries one proton, at low pH both groups do so, whereas at high pH neither group carries a proton. The pK values of the free enzyme that govern the proton release, 4.2 and 8.5, correspond to those that govern overall activity. Both from the experiments with methylmercuric iodide and from fluorescence measurements of methylmercuric papain, it was established that the imidazole group in the latter compound exhibits a pK of 5.4. Taking recent data into account, it was considered that the ion pair of thiolate anion and imidazolium cation, proposed by Polgar, is the best approximation to describe the charge distribution in the active centre and to explain the reaction mechanism.

Binding Sites↗
PubMed1977

Dimerization of papain induced by mercuric chloride and a bifunctional organic mercurial.

The bifunctional mercurial meso-1,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed.

4-Chloromercuribenzenesulfonate↗
PubMed1979

Deuterium isotope effects on papain acylation. Evidence for lack of general base catalysis and for enzyme--leaving-group interaction.

The experimental data presented in this paper comprise kinetic deuterium isotope effects on acylation of papain with various substrates when conducted in H2O and 2H2O. With alkyl esters of N-acylamino acids there is no or very little isotope effect, whereas with N-acylamino acid amides the ratio kappa H2O/kappa 2H2O is less than 1, i.e. there is an inverse isotope effect. Similarly, alkylation of papain with methyl bromoacetate exhibits no kinetic isotope effect, whereas for the analogous alkylation with bromoacetamide an inverse isotope effect is observed. It is concluded that (a) general base catalysis does not occur in the acylation of papain and (b) kinetic deuterium isotope effects can be affected substantially by interaction between the substrate leaving group and the enzyme, which has not been considered in previous mechanistic investigations.

Acylation↗