The structure and mechanism of action of papain

1970 ◽  
Vol 257 (813) ◽  
pp. 237-248 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Mechanism ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Preceding Paper ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Critical Survey ◽  
Cysteine Proteinases ◽  
Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

197 INHIBITION OF CYSTEINE PROTEINASES DURING IN VITRO OOCYTE MATURATION IMPROVES DEVELOPMENT OF PORCINE CUMULUS - OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 211
Author(s):  
A. M. Lichtenauer ◽  
L. D. Spate ◽  
R. S. Prather ◽  
J. A. Green
Keyword(s):  
Oocyte Maturation ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Developmental Potential

Biochemical differences exist between oocytes that give rise to viable blastocysts and oocytes that give rise to embryos that are developmentally compromised. For example, specific proteolytic enzymes (e.g. cathepsin B) are transcriptionally abundant in in vitro-matured bovine oocytes from prepubertal heifers that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus–oocyte complexes (COC) that represented both poor- and good-quality oocytes. Those reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development. This project sought to determine if similar results would be obtained in a porcine oocyte/embryo culture system. Inclusion of 10 and 20 μM E-64 in maturation medium was performed. Maturation rates of porcine COC in 20 μM E-64 were elevated compared to those incubated in 10 μM E-64 (74% vs 53%; P < 0.05) or without E-64 (55%; P < 0.05: N = 1750 oocytes tested). Successful maturation to metaphase II was based on the presence of a polar body and a uniform cytoplasm 44 h after follicular aspiration. Based on these preliminary results and the earlier bovine work, it was hypothesized that the E-64 was having little influence on normal oocytes, but was promoting maturation of low-quality oocytes, possibly those that were beginning to degenerate. Consequently, 20 μM of E-64 was added to the maturation media of COC segregated based on morphological characteristics of the oocytes. Good COC had a homogeneous cytoplasm and greater than 3 layers of cumulus cells; the COC were considered poor if they displayed a nonhomogeneous cytoplasm and 1 layer or less of cumulus cells, yet were still considered fertilizable. Without E-64, an increase in maturation was measured when good oocytes were compared to poor oocytes (52% vs 29%; P < 0.05: N = 1600). No significant differences in maturation were observed between good oocytes incubated in the presence or absence of E-64. Likewise, no significant differences were observed between poor oocytes incubated in the presence or absence of E-64. The percentage of maturation of good oocytes cultured in E-64 was significantly higher than that of poor oocytes cultured with E-64 (67% vs 43%; P < 0.05). Maturation with the inhibitor did not significantly affect the subsequent cleavage or blastocyst rates of embryos that arose from these oocyte groups after fertilization. These experiments suggest that inhibition of cysteine proteinases significantly promotes oocyte maturation, as was seen in previous bovine work. Our data did not support the hypothesis that cysteine proteinase inhibition was selectively improving maturation of poor oocytes within the pool. It remains possible that increased maturation in good oocytes is a result of cysteine inhibition on juvenile oocytes that morphologically appeared good and the effect was less on already degenerated oocytes that appeared poor. Differences between treatments were determined by ANOVA with post-test by Tukey's multiple comparison test.

scholarly journals Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro

Parasitology ◽  
2004 ◽  
Vol 130 (2) ◽  
pp. 203-211 ◽  
Author(s):  
G. STEPEK ◽  
D. J. BUTTLE ◽  
I. R. DUCE ◽  
A. LOWE ◽  
J. M. BEHNKE
Keyword(s):  
Mechanism Of Action ◽  
Cysteine Proteinase ◽  
Gastrointestinal Nematode ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Fruit Extract ◽  
Potent Effect ◽  
Kiwi Fruit ◽  
Heligmosomoides Polygyrus

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract, containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.

scholarly journals A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and −161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-α-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide catalysed by cathepsin B and of l-arginine 2-naphthylamide catalysed by cathepsin H.

1985 ◽  
Vol 227 (2) ◽  
pp. 521-528 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Catalytic Activity ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ion Pairs ◽  
Preceding Paper ◽  
Structural Features ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Cathepsin H ◽  
Ph Range

The pH-dependences of kcat, Km and kcat./Km for the hydrolysis at 25 degrees C at I 0.1 of L-arginine 2-naphthylamide catalysed by cathepsin H from bovine spleen were determined in the pH range approx. 4-8. The pH-dependences of these kinetic parameters were determined also for the hydrolysis at 25 degrees C at I 0.1 of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B (EC 3.4.22.1) from bovine spleen in the pH range 7-8, which extends the studies in acidic media reported by Willenbrock & Brocklehurst [(1984) Biochem. J. 222, 805-814]. These results are discussed and related to those from the reactivity-probe kinetics reported in the preceding paper [Willenbrock & Brocklehurst (1985) Biochem. J. 227, 511-519] and to known structural features present in rat liver cathepsins B and H and in papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). Consideration of the kinetic data leads to the suggestion that in the cysteine proteinases rearrangement of intimate S-/ImH+ ion-pairs in catalytic sites is brought about by a combination of field effects in the immediate vicinity of the ion-pair and consequences of protonic dissociation of a group with pKa 5-6 remote from the catalytic site. The contributions of the two types of effect seem to differ from enzyme to enzyme. Of the four cysteine proteinases considered, only cathepsin B exerts an absolute requirement for the proton-deficient form of a group with pKa 5-6 for catalytic activity. Protonic dissociation with pKa 5-6 enhances catalytic activity in cathepsin H and in actinidin and appears to have little or no effect in papain. Only cathepsin B lacks a polar or negatively charged side chain in the residue analogous to Asp-158 in papain, and this is suggested to account for its total dependence on a protonic dissociation remote from the catalytic site.

scholarly journals Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases

1999 ◽  
Vol 343 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Jenny LJUSBERG ◽  
Barbro EK-RYLANDER ◽  
Göran ANDERSSON
Keyword(s):  
Cysteine Proteinase ◽  
Bovine Milk ◽  
Enzymic Activity ◽  
Purple Acid Phosphatase ◽  
Kinetic Properties ◽  
Subunit Structure ◽  
Acid Phosphatases ◽  
Cysteine Proteinases ◽  
Ph Optimum ◽  
Enzyme Preparations

Purple acid phosphatases (PAPs) are binuclear acid metallohydrolases also referred to as tartrate-resistant acid phosphatases (TRAPs) or type 5 acid phosphatases. The cDNA sequences of TRAP/PAP enzymes from different species and organs indicate that these enzymes are translated as monomeric polypeptides of approx. 35 kDa, contrasting with the predominantly two-subunit structure observed in purified enzyme preparations. In the present study we have compared certain structural and enzyme-kinetic properties of recombinant rat PAP (monomeric) with those of the native rat bone TRAP/PAP enzyme (two-subunit), and examined effects on these parameters by cleaving the monomeric recombinant PAP with the serine proteinase trypsin or the cysteine proteinases papain or cathepsin B. Cleavage with trypsin resulted in a moderate activation of the recombinant enzyme and shifted the pH optimum to a slightly more basic value (5.0-5.5). Cleavage with papain resulted in complete activation and conferred similar properties to those of the bone PAP variant with regard to pH optimum (5.5-6.0) and sensitivity to reducing agents, as well as in the sizes of the subunits. Substrate specificity studies showed that the two-subunit bone PAP was considerably more active than the monomeric recombinant rat PAP towards a variety of serine-, threonine- and tyrosine-phosphorylated substrates. Of these substrates, bovine milk osteopontin seemed to be the most readily dephosphorylated substrate. In conclusion, the results suggest that the monomeric form of PAP represent a latent proenzyme with low enzymic activity towards both tyrosine- and serine/threonine-containing phosphorylated substrates. Besides being implicated in the catabolism of the extracellular matrix, members of the cysteine proteinase family might also exert a regulatory role in degradative processes involving the PAP enzymes by converting the newly synthesized PAPs to enzymically active and microenvironmentally regulated species.

scholarly journals 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

1986 ◽  
Vol 233 (1) ◽  
pp. 119-129 ◽  
Author(s):  
B S Baines ◽  
K Brocklehurst ◽  
P R Carey ◽  
M Jarvis ◽  
E Salih ◽  
...  
Keyword(s):  
Close Contact ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Resonance Raman ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Catalytic Site ◽  
Cysteine Proteinases ◽  
Ph Values ◽  
Covalent Chromatography

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 × 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.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Fractionation, Purification and Some Properties of Proteolytic Enzymes from Stem Bromelain

1971 ◽  
Vol 35 (9) ◽  
pp. 1419-1430 ◽  
Author(s):  
Yoshinobu Minami ◽  
Etsushiro Doi ◽  
Tadao Hata
Keyword(s):  
Proteolytic Enzymes ◽  
Stem Bromelain

Fractionation and Partial Characterization of the Proteolytic Enzymes of Stem Bromelain*

Biochemistry ◽  
1963 ◽  
Vol 2 (3) ◽  
pp. 476-481 ◽  
Author(s):  
Mohamed El-Gharbawi ◽  
John R. Whitaker
Keyword(s):  
Proteolytic Enzymes ◽  
Partial Characterization ◽  
Stem Bromelain

Changes in conformation and enzymic activity of stem bromelain in alkaline media

Biochemistry ◽  
1970 ◽  
Vol 9 (9) ◽  
pp. 1935-1938 ◽  
Author(s):  
Takashi Murachi ◽  
Mitsue Yamazaki
Keyword(s):  
Enzymic Activity ◽  
Alkaline Media ◽  
Stem Bromelain

scholarly journals Cystatins — Inhibitors of Cysteine Proteinases

1992 ◽  
Vol 3 (4) ◽  
pp. 307-332 ◽  
Author(s):  
Libuse A. Bobek ◽  
Michael J. Levine
Keyword(s):  
Common Ancestor ◽  
Molecular Level ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Diverse Group ◽  
Cysteine Proteinases ◽  
Future Directions ◽  
Cysteine Proteinase Inhibitors ◽  
Physicochemical And Functional Properties

The cystatin superfamily of proteins, derived from a common ancestor, is comprised of a diverse group of potent cysteine proteinase inhibitors and antibacterial/viral agents grouped into several families. This review concentrates on family 2 cystatins, namely, the human salivary cystatins and cystatin C. Emphasis is given to their physicochemical and functional properties at both the protein and the molecular level. The role of cystatins in disease processes, including those in the oral cavity, is also discussed. Finally, future directions for cystatin research in oral biology are presented.

Sign in / Sign up

Export Citation Format

Share Document