Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2''-depyridyl disulphide as a reactivity probe.

1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.     

The reaction of papain with Ellman''s reagent [5,5′-dithiobis-(2-nitrobenzoate) dianion]

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.     

Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.     

Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols

1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.     

Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe.

1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2'-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2'-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2'-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin.

The kinetics of the reactions of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) with the two-protonic-state reactivity probes 2,2'-dipyridyl disulphide, n-propyl 2-pyridyl disulphide and 4-(N-aminoethyl 2'-pyridyl disulphide)- 7-nitrobenzo-2-oxa-1,3-diazole (compound I) were studied over a wide range of pH. Differences between the reactivities of ficin and papain towards the cationic forms of the alkyl 2-pyridyl disulphide probes suggest that ficin contains a cationic site without exact analogue in papain, and the striking difference in the shapes of the pH-rate profiles for the reactions of the two enzymes with compound (1) suggests differences in the mobilities or dispositions of the active-centre histidine imidazole groups with respect to relevant hydrophobic binding areas. The evidence from reactivity-probe studies that the papain catalytic mechanism involves substantial repositioning of the active-centre imidazole group during the catalytic act does not apply also to ficin. If ficin contains an aspartic acid residue analogous to aspartic acid-158 in papain, the pKa of its carboxy group is probably significantly lower than the pKa of the analogous group in papain.     

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.     

Differences in the chemical and catalytic characteristics of two crystallographically ''identical'' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4.

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2'-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2'-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2'-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.     

A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.     

Supracrystallographic resolution of interactions contributing to enzyme catalysis by use of natural structural variants and reactivity-probe kinetics.

1. The influence on the reactivities of the catalytic sites of papain (EC 3.4.22.2) and actinidin (3.4.22.14) of providing for interactions involving the S1-S2 intersubsite regions of the enzymes was evaluated by using as a series of thiol-specific two-hydronic-state reactivity probes: n-propyl 2-pyridyl disulphide (I) (a 'featureless' probe), 2-(acetamido)ethyl 2'-pyridyl disulphide (II) (containing a P1-P2 amide bond), 2-(acetoxy)ethyl 2'-pyridyl disulphide (III) [the ester analogue of probe (II)] and 2-carboxyethyl 2'-pyridyl disulphide N-methylamide (IV) [the retroamide analogue of probe (II)]. Syntheses of compounds (I), (III) and (IV) are reported. 2. The reactivities of the two enzymes towards the four reactivity probes (I)-(IV) and also that of papain towards 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide (VII) (containing both a P1-P2 amide bond and an L-phenylalanyl side chain as an occupant for the S2 subsite), in up to four hydronic (previously called protonic) states, were evaluated by analysis of pH-dependent stopped-flow kinetic data (for the release of pyridine-2-thione) by using an eight-parameter rate equation [described in the Appendix: Brocklehurst & Brocklehurst (1988) Biochem. J. 256, 556-558] to provide pH-independent rate constants and macroscopic pKa values. The analysis reveals the various ways in which the two enzymes respond very differently to the binding of ligands in the S1-S2 intersubsite regions despite the virtually superimposable crystal structures in these regions of the molecules. 3. Particularly striking differences between the behaviour of papain and that of actinidin are that (a) only papain responds to the presence of a P1-P2 amide bond in the probe such that a rate maximum at pH 6-7 is produced in the pH-k profile in place of the rate minimum, (b) only in the papain reactions does the pKa value of the alkaline limb of the pH-k profile change from 9.5 to approx. 8.2 [the value characteristic of a pH-(kcat./Km) profile] when the probe contains a P1-P2 amide bond, (c) only papain reactivity is affected by two positively co-operative hydronic dissociations with pKI congruent to pKII congruent to 4 and (d) modulation of the reactivity of the common -S(-)-ImH+ catalytic-site ion-pair (Cys-25/His-159 in papain and Cys-25/His-162 in actinidin) by hydronic dissociation with pKa approx. 5 is more marked and occurs more generally in reactions of actinidin than is the case for papain reactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding.

1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2'-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2'-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.     

A kinetic and fluorimetric investigation of papain modified at tryptophan-69 and -177 by N-bromosuccinimide

A systematic study of the modification of papain (its thiol group protected as a disulphide with mercaptoethanol) by N-bromosuccinimide, showed that 2 molar equiv. modified tryptophan-69 and 4 molar equiv. modified tryptophan-69 and -177. The Michaelis parameters for the catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester by these modified enzymes were determined. The enzymic activity of the modified enzymes was not seriously impaired, but modification of tryptophan-177 raised the apparent pK(a) of the acidic limb of the pH profile by more than 1 pH unit for both k(cat.) and k(cat.)/K(m). The fluorescence spectra (excitation at 288nm) of the modified enzymes showed that tryptophan-69 contributed about 8% to the fluorescence intensity, whereas tryptophan-177 contributed about 46% at neutral pH. However, the contribution of tryptophan-177 was quenched at low pH and its fluorescence intensity showed sigmoidal pH-dependence, with an apparent pK(a) of 4.2. Histidine-159, which is in close contact with tryptophan-177, is considered to be the residue responsible for the fluorescence quenching. When tryptophan-177 was modified, presumably generating a less hydrophobic micro-environment, the apparent pK(a) determined kinetically was raised to about 5.4. By comparing the Michaelis parameters of native papain, papain modified at tryptophan-69 and papain modified at tryptophan-69 and -177 with N-benzyloxycarbonylglycylglycine amide and N-benzyloxycarbonylglycyltryptophan amide, tryptophan-69 and tryptophan-177 were shown to be structural features of the S(2) and S(1)' subsites respectively.     

A two-step procedure for purification of papain from extract of papaya latex

A method is presented for purifying papain from extracts of papaya latex. The procedure involves precipitation of the extract of papaya latex with sodium chloride followed by affinity chromatography of the redissolved precipitate. Precipitation of the protein from the latex extract is necessary to separate the papain from material which interferes with the binding of papain to the affinity column. During affinity chromatography, the affinity column is overloaded to insure absence in the final product of impurities which are capable of binding to the affinity column.The papain prepared by this procedure yielded an amino acid analysis and an N-terminal amino acid analysis expected for a sample of pure papain. No Met was detected on amino acid analysis nor was the presence of N-terminal residues other than He detected. On polyacrylamide disc gel electrophoresis at pH 4.3, papain prepared by the method described in this work was indistinguishable from crystalline papain which was prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Both disc gel patterns consisted of a single band and a trailing shadow which was less than 5% of the main band. In routine spectrophotometric assays, the specific activity toward N,α-benzoyl-l-arginine ethyl ester of papain prepared by the procedure described in this work was indistinguishable from crystalline papain prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Values of 24 sec^?1' and 15 mm were obtained from the turnover number and K_m for the papain-catalyzed hydrolysis of N,α-benzoyl-l-arginine ethyl ester at 25 °C, pH 6.00, $\text{Γ}\text{2 0.30}$ using a pH stat.     

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.     

Purification of cathepsin B by a new form of affinity chromatography.

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.     

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2''-dipyridyl disulphide as a thiol titrant and reactivity probe.

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.