Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain.

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.     

The electrostatic fields in the active-site clefts of actinidin and papain.

The active sites of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2) display different reactivity characteristics to probes targeted at the active-site cysteine residue despite the close structural similarity of their active sites. The calculated electrostatic fields in the active-site clefts of actinidin and papain differ significantly and may explain the reactivity characteristics of these enzymes. Calculation of electrostatic potential also focuses attention on the electrostatic properties that govern formation of the active-site thiolate-imidazolium ion-pair. These calculations will guide the modification of the pH-activity profile of the cysteine proteinases by site-directed mutagenesis.     

Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain

An accurate three-dimensional structure is known for papain (1.65 A resolution) and actinidin (1.7 A). A detailed comparison of these two structures was performed to determine the effect of amino acid changes on the conformation. It appeared that, despite only 48% identity in their amino acid sequence, different crystallization conditions and different X-ray data collection techniques, their structures are surprisingly similar with a root-mean-square difference of 0.40 A between 76% of the main-chain atoms (differences less than 3 sigma). Insertions and deletions cause larger differences but they alter the conformation over a very limited range of two to three residues only. Conformations of identical side-chains are generally retained to the same extent as the main-chain conformation. If they do change, this is due to a modified local environment. Several examples are described. Spatial positions of hydrogen bonds are conserved to a greater extent than are the specific groups involved. The greatest structural similarity is found for the active site residues of papain and actinidin, for the internal water molecules and for the main-chain conformation of residues in alpha-helices and anti-parallel beta-sheet structure. This was reflected also in the similarity of the temperature factors. It suggests that the secondary structural elements form the skeleton of the molecule and that their interaction is the main factor in directing the fold of the polypeptide chain. Therefore, substitution of residues in the skeleton will, in general, have the most drastic effect on the conformation of the protein molecule. In papain and actinidin, some main-chain-side-chain hydrogen bonds are also strongly conserved and these may determine the folding of non-repetitive parts of the structure. Furthermore, we included primary structure information for three homologous thiol proteases: stem bromelain, and the cathepsins B and H. By combining the three-dimensional structural information for papain and actinidin with sequence homologies and identities, we conclude that the overall folding pattern of the polypeptide chain is grossly the same in all five proteases, and that they utilize the same catalytic mechanism.     

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.     

Semiempirical and Raman spectroscopic studies of carotenoids

Semiempirical AM1 calculations have been carried out for beta-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of beta-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12'; 13,13'; 14,14'; 15,15'; 15, and 20,20' were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310-321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115-178; Spectrochim Acta 1996, 53, 381-392; Biochim Biophys Acta 1994, 1185, 188-196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins.     

Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.     

A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion.

1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.     

The proteolytic activities of chymopapain, papain, and papaya proteinase III

The three proteinases present in papaya latex: papain (EC 3.4.22.2) chymopapain and papaya proteinase III (EC 3.4.22.6), were standardized by active-site titration, and compared in proteolytic activity against azocasein, serum albumin and cartilage proteoglycan. The activities were all of the same order, although there were differences in pH dependence. SDS-polyacrylamide gel electrophoresis of the early products of digestion of albumin and phosphorylase a showed very similar patterns for the three papaya proteinases. Kinetic parameters for hydrolysis of benzyloxycarbonyl-phenylalanyl-arginyl-7(4-methyl)coumarylamide were determined for the three enzymes. Values for kcat/Km varied only within a factor of 2, but the individual constants were much higher for papain than for chymopapain and papaya proteinase III. In contrast to the results obtained with the synthetic substrate, the kinetic parameters for the initial hydrolysis of succinyl-albumin were very similar for the three papaya proteinases. This was consistent with their similar proteolytic activities in other assays.     

Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: crystallographic, mutagenesis and resonance Raman spectroscopic studies

FixL of Rhizobium meliloti (RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O(2) levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 A resolution. Based on the structural and spectroscopic analyses, we propose the O(2) sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O(2) and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O(2) and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O(2) and Fe-CN stretching frequencies were shifted from 575 to 570 cm(-1) (Fe-O(2)), and 504 to 499 cm(-1), respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O(2) sensing activity was destroyed, thus confirming our proposed mechanism.     

The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain.

1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.     

Isolation and characterization of papaya peptidase A from commercial chymopapain.

Chromatography on a column of SP-Sephadex shows that commercial chymopapain contains three components with proteolytic activity. Each behaves as a single protein upon rechromatography and electrophoresis on polyacrylamide gels. The major component, which represents 31% of the activity applied to the column and is the most basic protein, was identified as papaya peptidase A. This enzyme has no methionine and isoleucine on its N-terminus. Its molecular weight is about 24,000 as determined by sodium dodecyl sulfate polyacrylamide electrophoresis and sedimentation equilibrium centrifugation. Its fluorescence emission as a function of pH resembles that for unactivated papain. Reduction is required for full activity, and in general it is less active than papain against substrates such as casein, N-benzoyl-L-arginine ethyl ester, N-tosyl-L-arginine methyl ester, N-benzoyl-L-arginineamide, and N-benzoyl-DL-arginine p-nitroanilide. Of the other components isolated from crude chymopapain, the more acidic enzyme contains 20% of the activity applied to the column, has a molecular weight of about 25,000, and N-terminal residues of tyrosine and glutamic acid. The other enzyme represents 26% of the initial activity, has a molecular weight of about 28,000 and tyrosine on its N-terminus. Both proteins have a single residue of methionine per molecule. The more acidic component resembles chymopapain A, and the other enzyme is similar to chymopapain B.     

Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme.     

Differences between the electric fields of the catalytic sites of papain and actinidin detected by using the thiol-located nitrobenzofurazan label as a spectroscopic reporter group.

The catalytic-site thiol groups of papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were each labelled with the nitrobenzofurazan (Nbf) chromophore by reaction with 4-chloro-7-nitrobenzofurazan at pH 4.4. The electronic-absorption spectra of both labelled enzymes were determined in aqueous solution, in the pH ranges approx. 2-5 for S-Nbf-papain and approx. 3.3-8 for S-Nbf-actinidin, and for the latter also in 6 M-guanidinium chloride. The spectrum of S-Nbf-papain is characterized by lambda max. = 402 nm at pH 5 and by lambda max. = 422 nm at pH 2.18. The pH-dependent shift in lambda max. accompanies a pH-dependent change in A 430, the nature of which is consistent with its dependence on a single ionizing group with pKa 3.7. The spectrum of S-Nbf-actinidin is pH-independent in the pH range approx. 3.3-8 and is characterized by lambda max. = 413 nm. This absorption maximum shifts to 425 nm in 6M-guanidinium chloride. These results are discussed and related to those reported previously from studies on papain and actinidin with various reactivity probes. Despite the close similarity in the catalytic sites of papain and actinidin deduced from X-ray-diffraction studies, the considerable differences in their reactivity characteristics are mirrored by differences in their electric fields detected by the Nbf spectroscopic label. The microenvironment in the catalytic site of actinidin appears to favour the existence of ions significantly more than in the corresponding region in papain.     

Resonance Raman spectroscopic studies of adriamycin and copper(II)-adriamycin and copper(II)-adriamycin-DNA complexes

Characteristic resonance Raman spectra are observed on ionization of the phenolic groups in adriamycin. On the basis of these results, vibrational assignments for the Raman bands of adriamycin are reported. Distinct Raman spectra are observed for Cu(II)-adriamycin complexes at pH approximately 5 and pH approximately 13. The data indicate that at lower pH a bis complex of Cu(II) is formed, which transforms to a polymeric Cu(II) chelate at higher pH. Upon interaction of the metal-drug complex with calf thymus DNA at pH approximately 5, a ternary complex is formed in which the Cu(II)-complexed adriamycin is intercalated into DNA.