IV. Cysteine proteinases

1970 ◽  
Vol 257 (813) ◽  
pp. 231-236 ◽  
Keyword(s):  
Oxidation State ◽  
Proteolytic Enzyme ◽  
Enzyme Preparation ◽  
Chemical Nature ◽  
Enzymic Activity ◽  
Active Enzyme ◽  
Tropical Tree ◽  
Cysteine Proteinases ◽  
Sulphydryl Group ◽  
Sh Group

Papain (EC 3.4.4.10) is a proteolytic enzyme which is isolated from the Papaya, a common tropical tree. It is a sulphydryl enzyme and its SH group is required for enzymic activity. Papain as usually prepared (Kimmel & Smith 1954) contains only a small portion of active molecules. The majority of the molecules are inactive because their sulphydryl group is blocked. Part of the blocking is caused by disulphide formation with cysteine (Sluyterman 1967). This disulphide can be reduced by an excess of cysteine resulting in an active enzyme preparation. The free SH content never reaches 100% and is often not more than about 50% , so that we must distinguish between papain molecules with a reversibly and an irreversibly blocked SH group. The chemical nature of the irreversible blocking is not yet known. It might well be due to a higher oxidation state of the sulphur which cannot be reduced by an excess of cysteine (Glazer & Smith 1965).

scholarly journals Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1965 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
PJ Peterson ◽  
L Fowden
Keyword(s):  
Carboxylic Acid ◽  
Enzyme Preparation ◽  
Enzymic Activity ◽  
The Other ◽  
Asparagus Officinalis ◽  
Acid Activation ◽  
Phaseolus Aureus ◽  
Substrate Specificities ◽  
Imino Acids ◽  
Ammonium Sulphate Fractionation

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

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.

scholarly journals The chemical reactions of the haemagglutinins and neuraminidases of different strains of influenza viruses: I. Effect of reagents reacting with amino acids in the active centres

1969 ◽  
Vol 67 (2) ◽  
pp. 289-299 ◽  
Author(s):  
L. Hoyle
Keyword(s):  
Chemical Reactions ◽  
Protein Molecule ◽  
Enzymic Activity ◽  
Amide Group ◽  
Influenza Viruses ◽  
Active Centre ◽  
Neuraminidase Activity ◽  
Sh Group ◽  
Different Strains ◽  
Active Centres

SUMMARYStudies of the chemical reactions of the haemagglutinins and neuraminidases of eight strains of influenza viruses have been made by the use of chemical reagents reacting with chemically active groups in the protein molecule. The results indicate a close resemblance between the active centres of the haemagglutinins and neuraminidases in all the strains tested. In all cases the activities were unaffected by reagents reacting with the —SH group of cysteine, the —CH3S group of methionine, the amino group of lysine, the guanidyl group of arginine, or the indole ring of tryptophan. In all cases both the haemagglutinating and enzymic activities were reduced or destroyed by agents reacting with amide groups or reacting with both tyrosine and histidine.By the use of iodine under conditions in which tyrosine reacts but not histidine, and fluorodinitrobenzene under conditions in which histidine reacts more strongly than tyrosine, it was possible to detect a number of different active centres.(1) An active centre containing histidine and an amide group but not containing tyrosine was present in all the virus strains and was the only centre detectable in A1 and A2 strains. This type of centre appeared to possess both haemagglutinating and neuraminidase activity.(2) Active centres containing tyrosine and an amide group were detected in strains of A and B viruses. There was some evidence suggesting that tyrosine-containing centres were of two types: one possessing both haemagglutinating and enzymic activity while the other was a haemagglutinin without neuraminidase activity.The results could be explained by supposing that the presence of histidine in the active centre was essential for neuraminidase activity and that enzymically active tyrosine-containing centres also contained histidine, but that tyrosine could substitute for histidine, but that tyrosine could substitute for histidine in haemagglutinating centres.

scholarly journals A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen

1988 ◽  
Vol 256 (2) ◽  
pp. 433-440 ◽  
Author(s):  
R A Maciewicz ◽  
D J Etherington
Keyword(s):  
Cathepsin B ◽  
Specific Activity ◽  
Cathepsin L ◽  
Cross Reactivity ◽  
Enzymic Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Collagenolytic Activity ◽  
Cysteine Proteinases ◽  
Isoelectric Points ◽  
Multiple Charge

We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.

scholarly journals Corrigendum

Parasitology ◽  
2007 ◽  
Vol 134 (4) ◽  
pp. 607-607
Keyword(s):  
Active Site ◽  
Active Enzyme ◽  
Distilled Water ◽  
Cysteine Proteinases ◽  
Anthelmintic Efficacy ◽  
Order Of Magnitude ◽  
Papaya Latex ◽  
Published Paper

Stepek, G., Lowe A. E., Buttle D. J., Duce I. R. and Behnke J. M. (2007). Anthelmintic action of plant cysteine proteinases against the rodent stomach nematode, Protospirura muricola, in vitro and in vivo. Parasitology134, 103–112 (Published online 11 October 2006. doi:10.1017/S0031182006001302)The authors of the above article regret that several errors appear in their published paper:On Page 105: In the methods section entitled “In vivo assessment of anthelmintic efficacy of plant cysteine proteinases”, the sentence:‘Five grams of papaya latex were mixed with 8 ml of sterile distilled water (dH2O), filtered, and the amount of active enzyme present was measured, by active-site titration, to be 331 nmol’should read:‘Five grams of papaya latex were mixed with 8 ml of sterile distilled water (dH2O), filtered, and the amount of active enzyme present was measured, by active-site titration, to be 13·24 micromol’And the sentence:‘Each group of mice received a different treatment: 0·2 ml of papaya latex alone (containing 8 nmol active enzyme), …’should read:‘Each group of mice received a different treatment: 0·2 ml of papaya latex alone (containing 331 nmol active enzyme), …’On Page 110: In column 2, line 5, the sentence:‘The amount of active enzyme administered in each dose (8 nmol) is based …’should read:‘The amount of active enzyme administered in each dose (331 nmol) is based …’And on lines 10–15, the sentence:‘Assuming the volume of a mouse stomach to be 1 ml and the enzyme to be present throughout the stomach at equal dilution, the concentration of enzyme would be in the order of 8 microM, which is somewhat lower than the in vitro concentrations.’Should read:Assuming the volume of a mouse stomach to be 1 ml and the enzyme to be present throughout the stomach at equal dilution, the concentration of enzyme would be in the order of 330 microM, which is about an order of magnitude higher than effective in vitro concentrations. A possible reason for the …’

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