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At least 19 recordsLinked to original sources

PubMed1978

Mechanism of the reaction of papain with substrate-derived diazomethyl ketones. Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism.

The reactions of papain (EC 3.4.22.2) with substrate-derived diazomethyl ketones reported by Leary, Larsen, Watanabe & Shaw [Biochemistry (1977) 16, 5857--5861] are unusual in that (i) these reagents fail to react with low-molecular-weight thiols and (ii) the rate of reaction with the papain thiol group does not decrease to near-zero values across a pKa of 4 as the pH is decreased. Existing data are shown to suggest an interpretation involving neighbouring-group participation via transient thiohemiketal formation, rate-determining protonation by imidazolium ion and alkylation on sulphur via a three-membered cyclic transition state. Implications for (a) the difference in site-specificity exhibited by halomethyl ketones in their reactions with serine proteinases and cysteine proteinases and (b) stereoelectronic requirements in the mechanism of papain-catalysed hydrolysis are discussed. The possibility of two tetrahedral intermediates between adsorptive complex and acyl-enzyme is indicated.

Amino Acid Chloromethyl Ketones↗

PubMed1979

Evidence that binding to the s2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25-histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain.

A method is proposed by which site-specific reactivity probes that exhibit different reactivities in two ionization states can be used to detect association-activation phenomena that involve repositioning of acid/base groups in enzyme active centres. The pH-dependences of the apparent second-order rate constants (k) for the reactions of the thiol group of papain (EC 3.4.22.2) with a series of two-protonic-state reactivity probes are compared. The short-chain probes, 2,2'-dipyridyl disulphide and n-propyl 2-pyridyl disulphide, react at pH6 in adsorptive complexes and/or transition states with geometries that do not permit hydrogen-bonding of the pyridyl nitrogen atom with the active-centre imidazolium ion, as evidenced by the rate minima at pH6 and the rate maxima at pH4 provided by reagent protonation. Only when the probe molecule, e.g. 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole [compound(III)], contains a long hydrophobic side chain is the reaction characterized by maximal rates at about pH6, as in the acylation step of the catalytic act (at pH6, k(compound III)/k(2,2'-dipyridyl disulphide) approximately 100). It is proposed that this striking difference in profile shape may result from binding of the hydrophobic side chain of compound (III) possibly in the S(2)-subsite of papain, which promotes a change in catalytic-site geometry involving repositioning of the imidazolium ion of histidine-159 and hydrogen-bonding with the N atom of the leaving group, as has been postulated to occur in the acylation step of substate hydrolysis.

2,2'-Dipyridyl↗

PubMed1976

Effect of neuraminidase and papain treatment on lectin-induced agglutination of Novikoff tumor cells and assay of lectin receptor activity of the glycopeptides released from the cell surface by papain.

Lectins, plant proteins that bind specific saccharide determinants, have been utilized to examine the effect of neuraminidase digestion on the structure and/or expression of oligosaccharide moieties present at the periphery of Novikoff ascites hepatoma cells. Five lectins were utilized: concanavalin A (Con A), specific for alpha-D-manno- or alpha-D-glucopyranosyl residues; wheat germ agglutinin, specific for 2-acetamido-2-deoxy-D-glucopyranosyl residues; Ricinus communis agglutinin I (RCAI), specific for D-glucopyranosyl residues; R. communis agglutinin II (RCAII), specific for D-galacto- or 2-acetamido-2-deoxy-D-galactopyranosyl residues; and soybean agglutinin, specific for 2-acetamido-2-deoxy-D-galactopyranosyl residues. Neuraminidase treatment of Novikoff cells did not alter their agglutination by Con A or wheat germ agglutinin. Similar treatment produced only a 2-fold increase in their agglutination by RCAI but a 12-fold increase in their agglutination by RCAII, indicating that 2-acetamido-2-deoxy-D-galactopyranosyl residues become expressed upon neuraminidase treatment. This conclusion was confirmed by the observation that neuraminidase-treated Novikoff cells acquired agglutinability by soybean agglutinin. Binding studies using ferritin-conjugated RCAII indicated that neuraminidase treatment exposed cryptic cell surface receptors for RCAII. To ascertain the role of cell surface glycoproteins in lectin-induced agglutination of Novikoff cells, glycopeptides cleaved from the cell surface by papain were assayed for lectin receptor activity. The cell surface glycopeptides exhibited receptor activity for Con A, wheat germ agglutinin and RCAI but not for RCAII and soybean agglutinin. A cell surface macrosialoglycopeptide fraction, resolved by gel filtration and ion-exchange chromatography, possessed a major portion of the Con A and RCAI receptor activity.

Agglutination↗

PubMed1978

Conversion of the active-site cysteine residue of papain into a dehydro-serine, a serine and a glycine residue.

Photolysis of papain which had been inhibited with 2-bromo-2',4'-dimethoxyacetophenone regenerated papain, but also formed [deltaSer25]-papain (i.e. papain in which the active-site cysteine residue 25 was replaced by dehydroserine) via the intermediate dehydrocysteine analogue, [deltaCys25]-papain. Reduction with sodium borohydride gave [Ser25]papain. Both [Ser25]papain and [deltaSer25]-papain had binding properties similar to those of papain, but were devoid of enzymic activity. Their fluorescence properties were also investigated. Incubation of [deltaSer25]papain at pH 9.0 gave [Gly25]papain.

Acetophenones↗

PubMed1978

Respiratory hazards from papain.

Late-onset asthma occurred in a non-atopic worker in a factory where papain powder was packed. The patient had had 3 attacks of asthma since first being exposed to atmospheric papain dust. He gave a positive immediate reaction to skin-prick tests with solutions of papain which were appreciably weaker than solutions which caused no reaction in unexposed individuals. These results, together with his history and the fact that symptoms stopped when he was not exposed to papain, suggest that he had extrinsic allergic asthma caused by sensitisation to papain. Shortly after re-exposure to papain a worker in another factory died during an attack of asthma. Measures to prevent the inhalation of papain dust must be taken in factories where papain is handled, not only to avoid the proteolytic effects of the material but also to prevent workers from becoming sensitised.

Air Pollutants↗

PubMed1978

Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system.

1.2,2'-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.

2,2'-Dipyridyl↗

PubMed1975

Effect of papain on the interaction between human monocytes, erythrocytes, and IgG.

The mechanism by which papain detaches IgG-sensitized erythrocytes from the monocyte surface has been explored in an in vitro assay for the monocyte IgG receptor using red cells quantitatively sensitized with IgG anti-Rh D immunoglobulin. Papain treatment of IgG-sensitized erythrocytes diminished the ability of these cells to bind to the monocyte surface; however, treatment of erythrocytes with papain prior to sensitization with IgG did not inhibit binding, and at papain concentrations is greater than or equal to 38 mug/ml binding was enhanced. IgG receptor activity was not diminished by prior treatment of monolayer cells with papain and was enhanced with high concentrations of papain. These studies suggest that papain detaches erythrocytes from the monocyte surface by virtue of its proteolytic effect on IgG and not by an effect of papain on the D antigen of red cells or the IgG receptor on monocytes.

Animals↗

PubMed1977

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.

Enzymes, Immobilized↗

PubMed1976

Potentiometric determination of ionizations at the active site of papain.

The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.

Binding Sites↗

PubMed1979

A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods.

A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22 degrees C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515-531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and alpha-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis-Menten kinetics. The presence of the other enzymes results in an increase in the value of K(m) and a decrease in the catalytic-centre activity (k(cat.)) relative to the values for the catalysis by papain.

2,2'-Dipyridyl↗

PubMed1976

Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100.

gamma-Glutamyltranspeptidase is associated with the brush border membrane of kidney proximal straight tubule cells. It can be solubilized qualitatively by treatment with papain or Triton X-100. Neither procedure affects its catalytic activity but the two resulting forms of the enzyme differ considerably in their physical properties. The papain-solubilized transpeptidase is soluble in aqueous buffers and was purified 430-fold. It has an s20,w of 4.9 S, a Stokes radius of 36 A, and a calculated molecular weight of 69,000. It appears homogeneous by sedimentation equilibrium centrifugation (Mr=66,700). In contrast, the Triton-solubilized transpeptidase is soluble only in the presence of detergents and was purifed 300-fold. This form of the enzyme has a Stokes radius of 70 A but an s20,w of only 4.15 S. Aggregation of the enzyme just below the critical micelle concentration of Triton X-100 and its ability to bind 1.16 mg of Triton X-100-protein complex was calculated to be 169,000, but the glycoprotein portion of the complex is 52% of the total mass (87,000). The mass of Triton X-100 (82,000) is consistent with its reported micelle molecular weight. Treatment of the Triton-purified transpeptidase with papain or bromelain results in a form of the enzyme identical in all respects with the papain-purified enzyme. Both the Triton- and papain-purified transpeptidase exhibit two protein bands on sodium lauryl sulfate-polyacrylamide gel electrophoresis. The smaller subunits of the two forms appear identical (Mr=27,000), while the larger subunits of the Triton- and papain-purified enzyme have apparent molecular weights of 54,000 and 51,000, respectively. These data suggest that a peptide (3,000 to 19,000) in the larger subunit of gamma-glutamyltranspeptidase is responsible for its binding to Triton micelles and probably for holding the enzyme in the brush border membrane.

Animals↗

PubMed1977

Active water-insoluble derivatives of papain and other enzymes based on preformed diazonium-type supports.

Papain (EC 3.4.22.2) has been coupled to supports of titanium (IV) oxide and cellulose, which are particulate and pre-coated with diazotised 1,3-diaminobenzene, giving water-insoluble and stable derivatives which possess low proteolytic activity but high esterolytic activity. In addition the reversible binding of zinc (II) at the active site of papain has been exploited to inhibit protectively the enzyme during its linkage to the aforementioned supports, thereby yielding water-insoluble derivatives of papain having superior activity upon reactivation with EDTA. Application of the improved procedure of enzyme coupling to macroporous cellulose particles gave a water-insoluble derivative of papain having further enhanced proteolytic activity. Other properties of the water-insoluble derivatives of papain and of similarly prepared water-insoluble conjugates of urease (EC 3.5.1.5) and cholinesterase (EC 3.1.1.8) with cellulose are also reported.

Cellulose↗

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↗

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↗

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↗

PubMed1978

Identification of the functional ionic groups of papain by pH/rate profile analysis.

The pH dependence of papain catalysis was analyzed by a scheme which evaluates the kinetic contribution of both protonated and unprotonated species of functional groups involved in catalysis. Kinetic measurements were made at constant pH, without buffers, by automatic titration. The rate-determining step for papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester, determined by nucleophile competition, changed from acylation below pH 6.5 to mixed acylation-deacylation above pH 6.5. Kinetic analysis indicated that three prototropic groups governed the pH-specificity of alpha-N-benzoyl-L-arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of papain catalysis were functional with either two or three active-site protons. In base, a single functional ionic pathway is associated with an active site with only one proton. Pathways involving an unprotonated active site are catalytically inoperative in both acid and base. These results indicate that papain exhibits several catalytically functional ionic pathways. The results are discussed in terms of pKa assignments, and the mechanism of papain catalysis.

Arginine↗

PubMed1975

[Pulmonary function in papain induced emphysema in dogs (author's tranls)].

By intratracheal injection of the protease Papain to experimental animals parenchymal changes in the lung can be induced, that resemble human emphysema. Papain (dosage 26 to 112 mg, 1 to 4 injections) was given intratracheally to 8 bastard dogs, (weighing from 12.5 to 20 kg) during light general anesthesia. Pulmonary function was assessed in weekly intervals and related to morphologic changes in the lung. Static compliance of the lung and FRC measured during respiratory arrest were increased after papain, bronchial resistance, measured while artificially ventilated at constant pressure was also increased. Changes of static lung compliance and FRC were seen after the first administration of papain, but further increased with time of observation and after multiple doses of papain. Increase of resistance was not found before 5 weeks. At quiet breathing resistance was not increased at all. No significant changes were found of arterial pO2 and pCO2, pH, standard and actual bicarbonate, diffusion capacity for O2, tidal volume, minute ventilation and ventilatory rate. Morphological findings confirmed the changes described by others. Pulmonary function appears to be pathological at a time when morphology still seems to be normal. The question is discussed to what extent the model of experimental emphysema induced by proteolytic enzymes can contribute to the understanding of human pulmonary emphysema. Lung function in the course of experimental emphysema is compared with function in different clinical types of emphysema.

Airway Resistance↗