Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.     

Comparative resonance Raman spectroscopic and kinetic studies of acyl-enzymes involving papain, actinidin and papaya peptidase II.

Resonance Raman spectra are reported for a series of dithioacyl-enzymes involving actinidin (EC 3.4.22.14) and papaya peptidase II (the more basic monothiol cysteine proteinase of Carica papaya). The acyl groups are N-benzoylglycine and N-(beta-phenylpropionyl)glycine containing C = S or 13C = S at the ester function. Comparison of the data with those for the corresponding papain (EC 3.4.22.2) analogues [Storer, Lee & Carey (1983) Biochemistry 22, 4789-4796] allows us to define the conformation of the dithioacyl group in the catalytic site. In each case the dithioacyl group is bound in a single conformation known as conformer B, in which the glycinic nitrogen atom comes into close contact with the sulphur atom of the catalytic-site cysteine residue. For the N-(beta-phenylpropionyl)glycine dithioacyl-enzymes the torsional angles of the NH-CH2-C(= S) bonds assume values typical of an essentially relaxed non-strained state. However, for the N-benzoylglycine dithioacyl-enzymes there is evidence for a slightly perturbed conformer B, and the perturbation is most pronounced for N-benzoylglycine dithioacyl-actinidin. Values of k+2/Ks and k+3 for the reactions of papain, actinidin and papaya peptidase II with N-benzoylglycine and N-(beta-phenylpropionyl)glycine methyl thionoesters were obtained by a pre-steady-state kinetic study. Wide variation was found in k+2/Ks, but the values of k+3 are all similar. This general picture is supported by the results from a steady-state kinetic study of the reactions of the three enzymes with N-benzoyl-L-arginine-p-nitroanilide and with N-benzyloxycarbonyl-L-lysine p-nitrophenyl ester. The similarity of the values of k+3, together with the invariance of conformer B geometry at the P1 site, suggests that the chemistry of the deacylation process is highly conserved among these three cysteine proteinases.     

Identity of acyl group conformations in the active sites of papain and cathepsin B by resonance Raman spectroscopy

Resonance Raman spectroscopic data provide conclusive evidence for the existence of an acyl-enzyme intermediate during the reaction of a thionoester substrate, N-methyloxycarbonylphenylalanylglycine methyl thionoester (CH3OC(=O)-Phe-NHCH2C(=S) OCH3), with cathepsin B from porcine spleen. The resonance Raman spectrum of CH3OC(=O)-Phe-NHCH2C(=S)S-cathepsin B, where the thiol S is from the active-site cysteine residue, is compared to that of the corresponding papain acyl-enzyme. Within the limits of experimental error (+/-2 cm-1 for peak positions), there are no detectable spectral differences. Since the resonance Raman spectrum is sensitive to the torsional angles in the glycinic bonds and the cysteine linkages, the conformations are identical in those parts of the acyl-enzymes where chemical transformation occurs. A conformational analysis of the model compound CH3OC(=O)-Phe-NHCH2C(=S)SC2H5 demonstrates that the dithioacyl group in both dithioacyl-enzymes is present as a single population of a form known as conformer B. Conformer B is characterized by a small torsional angle about the glycinic NHCH2-CS(thiol) bond such that the nitrogen and S (thiol) atoms are in close contact. This conformer is widespread among the dithioacyl intermediates of plant cysteine proteinases, and it is apparent that the same chemistry is retained in a mammalian cysteine proteinase. Steady-state kinetic parameters are also reported for CH3OC(=O)-Phe-NHCH2C(=S)OCH3 reacting with papain and cathepsin B. The similarity of the Kcat values, 0.53 and 1.15 s-1, for papain and cathepsin B, respectively, provides further evidence for a conserved deacylation process.     

Definition of the site of reactivity of the ancestral protease of the papain type

Digestions of oxidized insulin B chain by the sulfhydryl proteases papain, chymopapain, papaya peptidase A, actinidin, bromelain, ficin and asclepains     

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)     

A comparison of esterolytic mechanisms of several sulfhydryl proteases

A comparison was made of the esterolysis reactions catalyzed by chymopapain, papaya peptidases A and B, several asclepains of both the A and B families, ficin, and bromelain. Michaelis parameters for a series of aryl mesyl glycinates were measured and plotted versus the relevant Hammett σ values. All enzymes exhibited the same response to the substrates used.     

Relaxed and perturbed substrate conformations in enzyme active sites: evidence from multichannel resonance raman spectra

A diode array based multichannel Raman spectrometer has made it possible to record complete, high quality, resonance Raman (RR) spectra of enzyme-substrate intermediates. The intermediates are dithioacylpapains in which the acyl group is either N-benzoylglycine or N-(beta-phenylpropionyl)glycine. RR data are reported for the unlabeled dithioacylpapains as well as for the intermediates labeled separately with ND, 15N, and 13C = S in the glycine residue. Comparison of the results for the dithioacylpapains with that of the corresponding labeled glycine ethyl dithioesters [Lee, H., Storer, A. C., & Carey, P. R. (1983) Biochemistry (preceding paper in this issue)] leads to the conclusion that for both substrates in the active site the dihedral angles in the glycine NH-C-C(= S) linkages assume an essentially relaxed type B conformation. Similarly, there is no evidence for distortion about the C(= O)-NH peptide bond which links the P1 and P2 sites on the substrate. However, for the N-benzoylglycine case there is evidence for some conformational distortion in the -S-C-C cysteine linkages. The present data favor a single homogeneous conformational population about the substrates' NH-C-C(= S) bonds in the native dithioacylpapains. However, below pH 3.0 the dithioacyl enzymes denature and the RR spectra of the 13C = S substituted species confirm that the conformational population reverts to the mixture of conformers A and B found for the corresponding ethyl dithioesters in solution.     

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.     

N-succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide as a sensitive substrate for assaying activity of cysteine proteinases

N-Succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide has been found to be a very sensitive chromogenic substrate for the assay of cysteine proteinase papain, ficin and bromelain. N-Succinyl-alanyl-S-benzylcysteine p-nitroanilide and N-succinyl-alanyl-alanyl-S-benzylcysteine p-nitroanilide are also suitable for this purpose. These substrates were hydrolyzed only very slightly or not hydrolyzed at all by trypsin.     

Surface Plasmon Resonance Imaging biosensor for cystatin determination based on the application of bromelain, ficin and chymopapain

A Surface Plasmon Resonance Imaging (SPRI) sensor based on bromelain or chymopapain or ficin has been developed for specific cystatin determination. Cystatin was captured from a solution by immobilized bromelain or chymopapain or ficin due to the formation of an enzyme-inhibitor complex on the biosensor surface. The influence of bromelain, chymopapain or ficin concentration, as well as the pH of the interaction on the SPRI signal, was investigated and optimized. Sensor dynamic response range is between 0-0.6 μg/ml and the detection limit is equal to 0.1 μg/ml. In order to demonstrate the sensor potential, cystatin was determined in blood plasma, urine and saliva, showing good agreement with the data reported in the literature.     

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.     

Amino acid and peptide derivatives of dimethyl 5-aminoisophthalate as fluorogenic substrates for proteinases

A number of amino acid and peptide derivatives of the fluorophore, dimethyl 5-aminoisophthalate have been synthesized, characterized and tested as substrates for the plant cysteine proteinases papain, ficin and bromelain. In every case, replacement of alanine by citrulline, in the position adjacent to the dimethyl 5-aminoisophthalate resulted in a higher rate of hydrolysis. The partly deprotected dipeptide derivative dimethyl phenylalanylcitrulline-5-aminoisophthalate was hydrolysed most rapidly of all the compounds tested, and on this basis may provide a useful substrate for the detection and quantitative assay of these enzymes.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: II. Inhibitors of subtilisin and trypsin

Two subtilisin inhibitors and two trypsin-chymotrypsin inhibitors were purified from seeds of Vigna unguiculata subsp. cylindrica. A third subtilisin inhibitor was partially purified. The subtilisin isoinhibitors were present in very small amounts in the seeds and the degree of purification of the three inhibitors was 20,000- to 48,000-fold. The purified inhibitors were found to be homogeneous on ultracentrifugation and polyacrylamide gel electrophoresis with or without dodecyl sulfate. The subtilisin inhibitors had no action on papain, ficin, chymopapain, bromelain, trypsin, chymotrypsin, or papain and the trypsin-chymotrypsin inhibitors were also inactive with other enzymes.     

Naturally occurring protein crystals in the potato : inhibitor of papain, chymopapain, and ficin

Protein crystals isolated from potato tubers were found to consist of a proteinase inhibitor active against the cysteine proteinases papain, chymopapain, and ficin. The molecular weight as determined by gel filtration at pH 4.3 or by gel electrophoresis in the presence of dodecylsulfate was 80 kilodaltons. When the inhibitor was evaluated at pH 8.4 in a linear concentration (4-30% polyacrylamide) under nondenaturing conditions, it appeared as two bands of approximately 320 to 350 kilodaltons indicating that the inhibitor forms tetrameric aggregates in neutral or weakly alkaline media, while the monomeric form predominates under acidic conditions. Gel filtration in the presence of varying amounts of papain suggested that the monomer combines with four papain molecules. The inhibitor contains no cystine.     

Comparative study on the active sites of ficin and papain by the spin labeling method

Ficin was alkylated with a series of haloacetamide spin labels with various distances between the spin probes and reactive groups. From the relation of these distances to the tau c values of the labels incorporated into protein, it was estimated that the depth of the active site hole of ficin is ca. 8 A. The results are somewhat different from those reported previously for papain (S. Nakayama et al. (1981) Biochem. Biophys. Res. Commun. 98, 471-475). Examination of the pH dependence of the ESR spectra for ficin and papain alkylated with an iodoacetamide or a maleimide spin label suggested that these enzymes have an amino acid residue of pKa 4 (probably a histidine residue) around the active site cysteine and that the active site conformations change at around pH 5.     

Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin

The cysteine proteinase inhibitor cystatin, from chicken egg white, bound with equimolar stoichiometry to the cysteine proteinases actinidin, chymopapain A, and ficin. The changes of near-ultraviolet absorption and fluorescence induced by the binding differed appreciably for the three enzymes, indicating that these spectral changes arise predominantly from aromatic residues in the proteinases. In contrast, the near-ultraviolet circular dichroism changes were similar for all three enzymes, supporting previous evidence that these changes originate mainly from the single tryptophan residue in cystatin, Trp-104. The pseudo-first-order rate constant for the binding increased linearly with the inhibitor concentration up to as high concentrations as could be measured for the three proteinases. This behavior is consistent with the complexes being formed by simple, bimolecular reactions, as was concluded previously for the reaction of cystatin with active and inactivated forms of papain. The second-order association rate constant varied only about 4-fold, from 2.2 X 10(6) to 9.6 X 10(6) M-1.s-1, for the three enzymes, the higher of these values being similar to that measured previously for the reaction with papain. These observations are consistent with the association rate being governed mainly by the frequency of collision between the binding areas of enzyme and inhibitor. All three cystatin-proteinase complexes dissociated to intact inhibitor, demonstrating reversibility. The dissociation rate constants varied about 20000-fold, from 4.6 X 10(-7) s-1 for ficin to 1.1 X 10(-2) s-1 for actinidin, reflecting substantial differences between the enzymes in the nature of the interactions with the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.     

Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates

To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A, and ficin, we synthesized N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester and compared its hydrolysis with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. It was found that the substitution of sulfur for the carbonyl oxygen hardly affected the second-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases [Asbóth, B., & Polgár, L. (1983) Biochemistry 22, 117-122], where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Therefore, and because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases.     

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.     

The catalytic mode of cysteine proteinases of papain (C1) family

The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.