Nuclear magnetic resonance studies on the pKa values and interaction of ionizable groups in bromelain inhibitor VI from pineapple stem

Bromelain inhibitor VI (BI-VI), a cysteine proteinase inhibitor from pineapple stem, is a unique double-chain molecule composed of two distinct domains A and B. In order to clarify the molecular mechanism of the proteinase-inhibitor interaction, we investigated the electrostatic properties of this inhibitor. The inhibitory activity toward bromelain was revealed to be maximal at pH 3-4 and the gross conformation to be stable over a wide range of pH. Based on these results, pH titration experiments were performed on the proton resonances of BI-VI in the pH range of 1.5-9.9, and pKa values (pKexp) were determined for all carboxyl groups and alpha-amino groups. The pKexp were also compared with theoretical values calculated from the NMR-derived structures of BI-VI. The electrostatic surface potential map constructed using the pKexp values revealed that BI-VI possesses continuous negatively charged and scattered positively charged regions on the molecular surface and both regions appear to serve for docking properly with a basic target enzyme. Furthermore, it was suggested that the ionic interaction of the inhibitor with the target enzyme is primarily important for the inhibition, which seems to involve some carboxyl groups in the inhibitor and a thiol group in the proteinase.     

Inhibition of cysteine proteinases by a protein inhibitor from potato

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.     

Isolation and Characterization of Two Forms of an Acidic Bromelain Stem Proteinase

Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.     

The cysteine proteinases of the pineapple plant.

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.     

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).     

Absolute side-chain structure at position 13 is required for the inhibitory activity of bromein

Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition.     

Cysteine proteinase activity in various developmental stages of Clonorchis sinensis: a comparative analysis

1. Cysteine proteinase activity in acidic extracts of various developmental stages of Clonorchis sinensis (metacercariae, 1-, 2-, and 3-month old worms) was examined. All the activities were maximum at acidic pH and showed inhibitor susceptibilities similar to the vertebrate cysteine proteinases. 2. Specific activity of cysteine proteinase(s) was highest in metacercariae with either CBZ-phe-arg-AFC or Azocoll as the substrate. The immature and mature worms had similar (but less than metacercariae) levels of activity. 3. A soluble cysteine proteinase with a native molecular weight of approximately 20,000 +/- 1414 was partially purified from 1-, 2-, and 3-month worms. The molecular weight of similar activity in metacercariae was approximately 32,000. 4. Results suggest developmental regulation of cysteine proteinase activity in the life cycle of C. sinensis.     

Native and acid-denatured L-lactate dehydrogenase are different in the lysosomal proteinases involving in their overall degradation

When native and (LDH) acid-denatured lactate dehydrogenase were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin alpha as well as by a general cysteine proteinase inhibitor, N-(L3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamid e (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin alpha-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.     

Cysteine proteinases from Schistosoma haematobium adult worms.

To identify and characterize cysteine proteinases from Schistosoma haematobium, lyophilized adult worms were homogenized, and enzymes were isolated and purified. From extracts prepared in acidic buffer, 3 putative cysteine proteinases were identified either directly or indirectly. The first proteinase (ShCP1) was identified by labeling with a radioiodinated inhibitor, Z-Tyr-Ala-CHN2, as a 35-kDa protein. However, it could not be detected by silver staining, amino acid sequencing, or by a monoclonal antibody specific for a similar molecule from Schistosoma mansoni. A second cysteine proteinase, ShCP2, was purified by gel filtration and dialysis. This 32-kDa molecule was thiol-dependent and was labeled with Z-Tyr-Ala-CHN2. The amino terminal amino acid sequence of ShCP2 showed remarkable similarity (up to 77%) to that of S. mansoni cathepsin B (SmCP2) as well as to mammalian cysteine proteinases. Both ShCP1 and ShCP2 reacted with polyclonal antibodies against S. mansoni, suggesting the existence of shared antigenic epitopes. A third activity, ShCP3, was identified as possibly a distinct proteinase based on its similarities to a 28-kDa cysteine proteinase from S. mansoni. This preliminary investigation demonstrates that the overall profile of cysteine proteinases in S. haematobium is very similar to that of S. mansoni.     

Electrostatic interactions in proteins: Calculations of the electrostatic term of free energy and the electrostatic potential field

The pH-dependence of the electrostatic energy of interactions between titratable groups is calculated for some well studied globular proteins: basic pancreatic trypsin inhibitor, sperm whale myoglobin and tuna cytochrome c. The calculations are carried out using a semi-empirical appraach in terms of the macroscopic model based on the Kirkwood-Tanford theory. The results are discussed in the light of their physicochemical and biological properties. It was found that the pH-dependence of the electrostatic energy correlates with the III–IV transition of cytochrome c. The electrostatic field of the cysteine proteinase inhibitor, cystatin, was calculated in two ways. In the first one, the electrostatic field created by the pH dependent charges of the ionizable groups and peptide dipoles was calculated using the approach proposed. In the second one, the finite-difference method was used. The results obtained by the two methods are in overall agreement. The calculated field was discussed in terms of the binding of cystatin to papain.     

Active-site geometry of proteinase K. Crystallographic study of its complex with a dipeptide chloromethyl ketone inhibitor

Proteinase K (EC 3.4.21.14) from the fungus Tritirachium album Limber is the most active known serine endopeptidase. The sequence of its 275-residue long polypeptide chain and its three-dimensional folding show a high degree of homology with the bacterial subtilisin proteases. Using difference Fourier methods, the binding mode of the synthetic carbobenzoxy-Ala-Ala-chloromethyl ketone inhibitor to the active site of proteinase K was determined. In several cycles of restrained least-squares, the enzyme-inhibitor complex was refined to a current R = 22% for 9400 X-ray diffraction data between 2.2 and 5.0 A resolution. The inhibitor is attached to proteinase K by two covalent bonds: one between the methylene carbon of the inhibitor and N epsilon 2 of the catalytic His 68, the other between the ketone carbon atom of the inhibitor and O gamma of the catalytic Ser 221. In addition, two hydrogen bonds donated by the peptide NH of Ser 221 and by the side chain NH2 of Asn 160 hold the hemiketal O- in the oxyanion hole. The peptide inhibitor is further hydrogen bonded to the proteinase polypeptide chain in a three-stranded antiparallel pleated sheet.     

Mutagenesis and kinetic studies of a plant cysteine proteinase with an unusual arrangement of acidic amino acids in and around the active site

We report the cloning, overexpression, kinetic analysis, and modeling of the tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3.4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.     

Cystatin S: a cysteine proteinase inhibitor of human saliva

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.     

Isolation and Characterization of Glutamyl Endopeptidase 2 from Bacillus intermedius 3-19

The culture filtrate of Bacillus intermedius 3-19 was used for isolation by chromatography on CM-cellulose and Mono S columns of a proteinase that is secreted during the late stages of growth. The enzyme is irreversibly inhibited by the inhibitor of serine proteinases diisopropyl fluorophosphate, has two pH optima (7.2 and 9.5) for casein hydrolysis and one at pH 8.5 for Z-Glu-pNA hydrolysis. The molecular weight of the enzyme is 26.5 kD. The K(m) for Z-Glu-pNA hydrolysis is 0.5 mM. The temperature and pH dependences of the stability of the proteinase were studied. The enzyme was identified as glutamyl endopeptidase 2. The N-terminal sequence (10 residues) and amino acid composition of the enzyme were determined. The enzyme hydrolyzes Glu4-Gln5, Glu17-Asp18, and Cys11-Ser12 bonds in the oxidized A-chain of insulin and Glu13-Ala14, Glu21-Arg22, Cys7-Gly8, and Cys19-Gly20 bonds in the oxidized B-chain of insulin.     

Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani

1. During development of Paragonimus westermani, larvae develop during migration within the host, and adult worms feed on pulmonary tissues, causing significant pathology in the mammalian host. In this report acidic extracts of various developmental stages (metacercariae and worms at one, two and three months of development) were examined for cysteine proteinase activity. 2. A soluble thiol-dependent proteinase activity with a native molecular weight of approximately 20,000 was isolated and partially purified. 3. The enzymes purified from the various developmental stages of the parasite had maximal activity at acidic pH and showed inhibitor susceptibilities similar to the vertebrate acidic cysteine proteinases. 4. Enzymatic activity was stable at pH 5.0 for at least two days when stored at 4 degrees C. 5. It is suggested that these enzymes may be involved in the nutrition of these parasites and/or during penetration and lysis of the tissues.     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.     

Acidic cysteine proteinase inhibitor in seminal plasma

A cysteine proteinase inhibitor with acidic isoelectric point (pI = 4.7-5.0) was found in human seminal plasma. Its apparent molecular mass is 16 kDa. It inhibits cysteine proteinases like ficin, cathepsin H, cathepsin B and papain. The inhibitory activity of seminal plasma against ficin is almost the same as that of human serum.     

Native and Acid-denatured L-Lactate Dehydrogenase Are Different in the Lysosomal Proteinases Involving in Their Overall Degradation

When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.     

Tight binding inhibitors of scytalone dehydratase: effects of site-directed mutations

We explore the use of site-directed mutations of scytalone dehydratase to study inhibitor binding interactions. The enzyme is the physiological target of new fungicides and the subject of inhibitor design and optimization. X-ray structures show that potent inhibitors (K(i)'s approximately 10(-)(11) M) interact mostly with 11 amino acid side chains and, in some cases, with a single backbone amide. Fifteen site-directed mutants of the 11 enzyme residues were prepared to disrupt enzyme-inhibitor interactions, and inhibition constants for 13 inhibitors were determined to assess changes in binding potencies. The results indicate that two of the six hydrogen bonds (always present in X-ray structures of native enzyme-inhibitor complexes) are not important for inhibitor binding. The other four hydrogen bonds are important for inhibitor binding, and the strength of the individual bonds is inhibitor-dependent. Inhibitor atoms remote from the hydrogen bonds influence their strength, presumably by effecting small changes in inhibitor orientation. Several hydrophobic amino acid residues are important recognition elements for lipophilic inhibitor functionalities, which is fully consistent with X-ray structures determined from crystals of enzyme-inhibitor complexes grown at neutral pH but not with those determined from crystals grown under acidic conditions. This study of mutant enzymes complements insights from X-ray structures and structure-activity relationships of the wild-type enzyme for refining views of inhibitor recognition.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.