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PubMed1989

Mode of binding of E-64-c, a potent thiol protease inhibitor, to papain as determined by X-ray crystal analysis of the complex.

The three-dimensional structure of the E-64-c-papain complex has been determined by X-ray crystal analysis at 2.5 A resolution (conventional R = 26.9%). The structure determined indicates that: (i) the C2 atom of the oxirane ring of E-64-c is covalently bound by the S gamma atom of Cys-25 of papain; (ii) this covalent bond formation results in a configurational conversion of the oxirane C2 atom from the S- to the R-form; and (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the specific interaction of the E-64-c molecule with papain.

Binding Sites↗
PubMed1993

X-ray crystallographic structure of a papain-leupeptin complex.

The three-dimensional structure of the papain-leupeptin complex has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S' subsites; (ii) the 'carbonyl' carbon atom of the inhibitor is covalently bound by the Cys-25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the 'carbonyl' oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln-19 and Cys-25.

Amino Acid Sequence↗
PubMed1992

Occurrence of an allosteric transition in the modification of papain with L-1-acetyl-2,3-dihydropyrrolo[2,3-b]-indole-2-carboxamide.

When papain was reacted with L-1-acetyl-2,3-dihydropyrrolo[2,3-b]indole- 2-carboxamide at pH 8.0, inactivation occurred accompanied by modification of Cys-25 in the active site. Plots of pseudo first-order rate constants against the reagent concentrations yielded an anomalous sigmoidal curve, suggesting that papain responded to this reagent in an allosteric manner. This is supported by the fact that the presence of a moderate concentration (a twenty-fold molar excess) of N alpha-acetyl-L-tryptophanamide over papain accelerated the inactivation.

Amino Acids↗
PubMed1984

Structure of papain refined at 1.65 A resolution.

Papain is a sulfhydryl protease from the latex of the papaya fruit. Its molecules consist of one polypeptide chain with 212 amino acid residues. The chain is folded into two domains with the active site in a groove between the domains. We have refined the crystal structure of papain, in which the sulfhydryl group was oxidized, by a restrained least-squares procedure at 1.65 A to an R-factor of 16.1%. The estimated accuracy in the atomic co-ordinates is 0.1 A, except for disordered atoms. All phi/psi angles for non-glycine residues are found within the outer limit boundary of a Ramachandran plot and this provides another check on the quality of the model. In the alpha-helical parts of the structure, the C = O bonds are directed more away from the helix axis than in a classical alpha-helix, leading to somewhat longer hydrogen bonds, 2.98 A, compared to 2.89 A. The hydrogen-bonding parameters and conformational angles in the anti-parallel beta-sheet structure show a large diversity. Hydrogen bonds in the core of the sheet are generally shorter than those at the more twisted ends. The average value is 2.91 A. The hydrogen bond distance Ni+3-Oi in turns is relatively long and the geometry is far from linear. Hydrogen bond formation, therefore, is perhaps not an essential prerequisite for turn formation. Although the crystallization medium is 62% (w/w) methanol in water, only 29 out of 224 solvent molecules can be regarded with any certainty as methanol molecules. The water molecules play an important role in maintaining structural stability. This is specially true for internal water. Twenty-one water molecules are located in contact areas between adjacent papain molecules. It seems as if the enzyme is trapped in a grid of water molecules with only a limited number of direct interactions between the protein molecules. The residues in the active site cleft belong to the most static parts of the structure. In general, disorder in atomic positions increases when going from the interior of the protein molecule to its surface. This behavior was quantified and it was found that the point of minimum disorder is near the molecular centroid.

Amino Acid Sequence↗
PubMed1993

Effects of papain on the agglutination of canine red cells with serum autoantibodies.

The papain test, which detects incomplete anti-red blood cell (RBC) autoantibodies in serum, was positive in 12 out of 16 anaemic dogs. Positive results were significantly correlated (chi 2 = 11.1, P < 0.001) with increased levels of RBC-bound immunoglobulin, but in three of these cases it was considered that a diagnosis of autoimmune haemolytic anaemia could not be justified. Furthermore, enzyme-linked antiglobulin test measurements of RBC-reactive serum IgG were increased in only three of the dogs with a positive papain test. Papainised canine RBC did not consistently take up more serum IgG than untreated cells in an indirect enzyme-linked antiglobulin test. However, the zeta potential of the RBC was reduced after enzyme treatment, and electrophoretic analysis revealed that glycophorins, which bear a strong negative charge, were cleaved from the cell membrane. It is concluded that a positive papain test alone is not reliable in the diagnosis of canine autoimmune haemolytic anaemia and that the enzyme increases the agglutinability of RBC by reducing the mutually repulsive electrostatic forces between the cells, rather than by increasing the amount of autoantibody bound.

Anemia↗
PubMed1976

Effect of papain-induced emphysema on the distrubtion of pleural surface pressure.

Distribution of transpulmonary pressure (Ptp) at FRC, during spontaneous breathing, and during inflation of the relaxed respiratory system was studied in the supine, lateral and head-up postures after papain had been injected intratracheally into dogs. Functional and morphological changes of the lung resembling those of panlobular emphysema occurred in papain-treated dogs. In all postures the relationship between lung height and Ptp at FRC was steeper and shifted to the left of that obtaining for normal dogs. During artifical inflation changes of Ptp were larger in the dependent than in the upper lung and the vertical gradient of Ptp eventually disappeared, as observed in normal dogs. During spontaneous breathing changes of Ptp were uniform in the horizontal postures, but larger over the upper regions in the head-up posture; whereas they are uniform in all postures in normal dogs. From local Ptp values and lung P-V curve, distribution of specific lung volumes at FRC and ventilation during spontaneous breathing were assessed for both normal and papain-treated head-up dogs: the results agreed with those obtained using radioactive gases in normal and in elderly man or emphysematous subjects with no apparently localized lesions.

Animals↗
PubMed1980

Non-invasive lung function tests in rats with progressive papain-induced emphysema.

Non-invasive plethysmographic methods for measurement of thoracic gas volume (TGV), airway resistance (Raw) and phase difference (PD) between flow rate at the nose and mean alveolar pressure were adapted for use in anesthetized rats. Thirty-six male Sprague-Dawley rats (200-250 g), were treated in groups of six by a single transoral, intratracheal instillation of 2 mg . kg-1 or 4 mg . kg-1 of papain in saline, or left untreated as controls. Measurements of TGV, Raw, and PD were made before treatment and at 4-day intervals after treatment for 8 or 16 days. TGV increased 65 percent by the fourth day and showed no change beyond the eighth day. Raw (0.4 +/- 0.04 cm H20 . ml-1 . sec) was not different from that of the controls. PD did not change significantly from the pretreatment value (4.7 +/0 0.41 degrees). The TGV, Raw, and PD responses were similar in the 2 mg . kg-1 and 4 mg . kg-1 papain treated rats. The TGV results suggest that the destructive effects of papain on the lungs are complete by the eighth day. The Raw results indicate that the airways are unaffected. The PD data are consistent with theoretical modeling studies for uneven distribution of compliances but neglible inequality of peripheral resistances.

Airway Resistance↗
PubMed1990

Papain-catalysed synthesis of dipeptides: a novel approach using free amino acids as nucleophiles.

For the first time, papain-catalysed synthesis of peptide bonds was successfully carried out using free amino acids as nucleophiles. In kinetically controlled experiments employing pH-Stat-mode, the ester substrates Z-Ala-OMe and Z-Gly-OMe were coupled with alanine, glutamine, and Cys(Acm)-OH, respectively. Under optimized reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 10 mumol substrate mg-1 papain), the peptide yields ranged from 17% to 79%, depending on the structure of the amino and/or carboxyl component. The peptides formed were not hydrolysed under the chosen reaction conditions. With Z-Gly-OMe as the ester substrate, formation of the dipeptide was both rapid and high yielding. Papain-catalysed formation of peptide bonds applying free amino acids as nucleophiles might serve as an economic and easily manageable approach for the synthesis of short-chain peptides to be used in clinical nutrition.

Amino Acid Sequence↗
PubMed1993

Influence of polyhydroxylic cosolvents on papain thermostability.

Papain thermostability was studied, and non-first-order deactivation kinetics were observed. The results obtained were analyzed by a two-step series-type deactivation model involving the native and active enzyme, an active intermediate enzyme state, and a final inactive state, with excellent agreement. The influence of different polyhydroxylic cosolvents (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of papain at 60 degrees C was also studied. Analysis of the results by the assayed model showed that the main protective effect of cosolvents was observed in the second step of the deactivation profile. The results obtained were analyzed as a function of both the thermodynamic parameters and a protective effect, defined as the ratio of papain half-lives (with and without cosolvents) for the second deactivation step, showing in both cases an important stabilizing effect of these cosolvents on the enzyme. The overall protective effect of cosolvents was also related simultaneously to their concentration and their water activity-depressing power.

Biotechnology↗
PubMed1987

Enthalpy and entropy of hippuraldehyde hydration and binding to papain.

In aqueous solution hippuraldehyde, PhCONHCH2CHO, is approx. 10-times more extensively hydrated than simple aliphatic aldehydes. Studies of the temperature dependence of the NMR spectrum of hippuraldehyde and its hydrate in 2H2O indicate that the dehydration is endothermic (delta H = 3.9 +/- 0.9 kcal/mol), but entropically favored (delta S = 7.9 +/- 1.9 cal/mol per deg). For simple aliphatic aldehydes the corresponding dehydration is slightly more endothermic, but somewhat more favorable entropically. Fluorescence titration studies show that Kd for the hemithioacetal adduct of hippuraldehyde with papain-SH (1.2 microM at 298 K) is approx. 3000-times smaller than that estimated for simple thiols. Dissociation of hippuraldehyde from papain is endothermic by 18.1 kcal/mol, and this is only partly offset by the favorable entropy change of 33.7 cal/mol per deg. Thus, at least in thermodynamic terms, the binding of this putative 'transition-state analog' to papain is not unlike the binding of other small ligands to the active site of chymotrypsin.

Hippurates↗
PubMed1989

Isosteric and non-isosteric modification of carboxyl groups of papain.

Guanidinated mercuri-papain (Gu-papain) was reacted with N-ethylbenzisoxazolium tetrafluoroborate at pH 4.2, 0 degree C, to yield highly reactive N-ethylsalicylamide esters. On varying the amount of reagent applied 2.5-10 carboxyl groups were modified. Appropriate plotting of the data indicated that all 12 groups exposed in the X-ray structure were modified to an extent of 80% in the final preparation, concomitant with a similar loss of activity towards N alpha-benzoyl-L-arginine ethyl ester. The preparations regained complete activity on saponification of the ester groups and removal of some oligomeric material by gel filtration. Considerable activity was recovered when the ester groups were completely replaced by amide groups by subjecting the esters to ammonolysis in 2 M ammonium acetate/ammonia (pH 9.2). The final preparation, after gel filtration, exhibited Km = 57 +/- 1 mM and kcat = 26 +/- 0.2 s-1 towards BAEE (native papain Km = 18 mM and kcat = 26 s-1). It may be concluded that replacement of a bulky modifying group by an isosteric one may cause considerable recovery of activity, emphasizing the importance of isostericity in suppressing the ionizing ability of ionizable groups; furthermore, that a large shift in overall charge, caused by amidation of all accessible carboxyl groups, does not affect the catalytic steps. The absence of effect of side-chain charges on the ion pair in the active site is briefly discussed.

Ammonia↗
PubMed1992

Contributions to the S'-subsite specificity of papain.

The product ratio was analyzed for the papain-catalyzed acyl transfer from the specific acyl donor Mal-Phe-Ala-OEtCl to various nucleophilic amino components, ranging from amino acid amides to tripeptide amides. The data obtained are discussed in terms of binding specificity. From the structure-activity relationships for the S'1-P'1 interaction it follows that only three methyl(ene) groups can be accommodated in the S'1 subsite. Hydrophilic side chains are bound better to S'1 than indicated by their hydrophobicities. Negatively charged amino components are inefficient deacylating agents. However, there was no evidence for electrostatic contributions to the nucleophile binding. Amino components with bulky hydrophobic amino acid residues in the P'2 and in the P'3 position, respectively, are preferentially bound to Mal-Phe-Ala-papain. The results of this study can be applied to the planning of papain-catalyzed peptide synthesis reactions.

Acyltransferases↗
PubMed1994

Skeletal muscle myosin regulatory light chains conformation affects the papain cleavage of A1 light chains.

In the present study, the influence of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (RLC) on accessibility of myosin and heavy meromyosin alkali light chains (A1) for papain digestion was investigated. The properties of native and papain treated myosin and heavy meromyosin were compared. Exchange of magnesium ions bound to RLCs for calcium ions accelerates the digestion of A1 in the presence of ATP in dephosphorylated myosin, heavy meromyosin, acto-myosin and the acto-heavy meromyosin complex. In the absence of ATP the exchange of magnesium ions bound to RLCs for calcium ions delays the digestion of A1 in the acto-myosin complex. Myosin and heavy meromyosin having shortened A1 by papain cleavage shows decreased K(+)-ATPase and increased actin binding ability in the presence and absence of ATP. The cooperation of RLC and A1 with heavy chains in the changes of structural organization of myosin head during muscle contraction is discussed.

Animals↗
PubMed1995

Effects of ligand homologation and ligand reactivity on the apparent kinetic specificity of papain.

Papain, a prototype cysteine proteinase, shows a pronounced kinetic preference for substrates and inhibitors based on the Ac-L-Phe-Gly-structural motif. Replacing the L-Phe at position P2 with D-Phe, or with a less hydrophobic residue such as Leu or Met, results in decreases of substrate or inhibitory activity of up to 400-fold. In this study we examined the effect of homologating the P1 glycine moiety to beta-alanine in the context of specific ester and amide substrates, peptidyl nitrile and -aldehyde transition state analog inhibitors, and peptidyl Michael acceptors as irreversible affinity labels. Papain discriminates extremely strongly (i.e., from 1000-fold to > or = 29,000-fold) against the 'homologs' based on beta-alanine at P1 compared to 'analogs' based on glycine at P1. However, with highly reactive ligands such as p-nitrophenyl esters, homolog/analog discrimination is greatly reduced (i.e., < or = 10-fold). These observations are interpreted in terms of (1) cooperativity between several non-covalent enzyme-ligand interactions and the covalent interaction of the ligand P1 moiety with Cys-25 of papain, (2) the decreased ability of homologs to utilize these cooperative interactions optimally because of their extended size, and (3) a decrease in the importance of the cooperative interactions as the intrinsic chemical reactivity of the ligand increases. Some implications of this analog vs. homolog discrimination for peptidyl disulfide and peptidyl chloromethane probes of protease specificity and mechanism are discussed.

Aldehydes↗
PubMed1987

Further studies of plasma protease inhibitors in the hedgehog, Erinaceus europaeus; collagenase, papain and plasmin inhibitors.

Hedgehog plasma was separated by gel filtration on Sephacryl S-200, the fractions resolved by electrophoresis and the electrophoretograms characterized for collagenase, papain and plasmin inhibiting activities with the high mol. wt substrate casein. The three inhibitors previously identified as alpha 2-, alpha 2-beta- and beta-macroglobulins were found to inhibit all three proteases. These were the only collagenase inhibitors found in plasma. Hedgehog alpha 2-chymotrypsin inhibitor and beta-protease inhibitor were both found to also inhibit papain. Three new inhibitors specific for papain (gamma-, alpha 2- and alpha 1-cysteine protease inhibitors) and one for plasmin (alpha 2-antiplasmin) were also found, bringing the number of protease inhibitors in hedgehog plasma to 14. Immunological cross-reactivity as studied by immunoelectrophoresis showed homology between hedgehog alpha 2-macroglobulin and rat murinoglobulin I and between hedgehog alpha 2-antithrombin and rat antithrombin III.

Animals↗
PubMed1991

Reversible binding of peptide aldehydes to papain. Structure-activity relationships.

The hydration of eleven peptide and hipuryl aldehydes has been measured as a function of temperature by means of NMR spectroscopy. In all cases the aldehydes were strongly hydrated (i.e., 90-95%) in aqueous solution. Dehydration of the hydrates was strongly endothermic, but this was partly offset by a positive entropy for dehydration. The binding of the aldehydes to papain was measured by fluorescence titration, and from these data dissociation constants for the hemithioacetal enzyme adducts were derived. Binding of N-Ac-L-PheNHCH2CHO (1) was particularly tight (Kd,corr = 0.00043 micro M) whereas that of its D-enantiomer (2) was 300-fold weaker (Kd,corr = 0.129 microM). The binding constants of the eleven aldehydes correlated with those for the reversible covalent binding of the analogous nitriles according to the equation log Kd(CHO) = -2.687 +/- 1.016 log K d(CN) (r = 0.99), lending support to previous suggestions that both peptide aldehydes and peptide nitriles behave as transition-state- or reactive intermediate analogs for papain. This finding is particularly striking in view of the obvious differences in hybridization (sp2 vs. sp3) and geometry (trigonal vs. tetrahedral) at the reactive P1 carbon center in their covalent adduct forms (thioimidate ester vs. hemithioacetal, respectively). A model for the binding of substrates, their transition states and analogs thereof is proposed. A key feature of the model is an obligatory covalent (or developing covalent) interaction between Cys-25-SH and the carbonyl or equivalent carbon of P1, augmented by intermolecular P1NH--OC(Asp-158), P2CO--HN(Gly-66) and P2NH--OC(Gly-66) hydrogen bonds and a hydrophobic P2-S2 interaction. The latter three interactions are optimum or nearly optimum when P2 is a hydrophobic L-amino acid with an N-acyl substituent. Data presented suggest that hippuryl derivatives are relatively non-specific substrates or inhibitors for papain and, consequently, are of diminished value as probes for binding and catalytic studies.

Aldehydes↗
PubMed1992

Induction of cystatin S in rat submandibular glands by papain.

1. Papain (a cysteine proteinase) were administered into the oral cavity of rats twice daily for 5 days. This treatment caused a dramatic increase in the level of cystatin S (a cysteine proteinase inhibitor belonging to family 2 of cystatin superfamily) in enlarged submandibular glands. 2. Immunochemical analysis using antibody against rat cystatin S and electrophoretic analysis confirmed that the protein induced by papain was identical to that induced by isoproterenol. 3. Induction of the cystatin S in the submandibular glands by oral administration of papain suggested a biological response which plays a role in preventing injury exogenous proteinase.

Animals↗
PubMed2005

Loosely packed papain prosegment displays inhibitory activity.

Most protease prosegments are co-synthesized at the N-termini of cysteine proteases and are involved in folding assistance, inhibition, and activation of their mature enzymes. By using circular dichroism, UV-difference and fluorescence spectroscopies, we studied the thermal unfolding of papain prosegment. The transition seems to be two-state and reversible, with an unfolded state prone to aggregation. Unfolding thermodynamic parameters obtained show low values both for deltaH(Tm) and deltaCp(U), indicative of a loosely packed three-dimensional conformation for the prosegment at near-neutral pH conditions. In spite of these results, fluorescence experiments demonstrate that papain prosegment is able to recognize and inhibit its cognate protease. An acid medium induces a molten globule-like state without intermediates, which in turn undergoes an irreversible thermal unfolding. Our results suggest that papain prosegment has a high degree of conformational flexibility, with the ability to form not only a molten globule-like structure in activating conditions, but also requiring an induced fit in order to be functional as inhibitor.

Animals↗