scholarly journals Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Brønsted coefficient (βnuc.) for the reactions of thiolate anions with 2,2′-dipyridyl disulphide may be decreased by reagent protonation

1980 ◽  
Vol 189 (1) ◽  
pp. 189-192 ◽  
Author(s):  
K Brocklehurst ◽  
B S Baines ◽  
M S Mushiri
Keyword(s):  
Carica Papaya ◽  
Cysteine Proteinases ◽  
Active Centre ◽  
Pka Values ◽  
Nitrobenzoic Acid ◽  
Specific Reactivity ◽  
Ph Range ◽  
Thiolate Anions ◽  
Active Centres

The active centres of chymopapains A and B (jointly designated EC 3.4.22.6) and papaya (Carica papaya L.) peptidase A were investigated by using 2,2′-dipyridyl disulphide and 5,5′-dithiobis-(2-nitrobenzoic acid) as thiol-specific reactivity probes. Whereas the first active-centre pKa values for chymopapain B and papaya peptidase A are less than 5, is as the case for papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), that for chymopapain A is about 6.8. The reason why the reactions of thiols of pKa approx. 6.5 with 2.2′-dipyridyl disulphide are essentially pH-independent in the pH range around the thiol pKa is delineated. The value of the Brønsted coefficient (beta nuc.) for the reactions of thiolate ions with the 2,2′-dipyridyl disulphide monocation appears to be smaller than its value for the corresponding reactions with the neutral disulphide.

scholarly journals 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

1983 ◽  
Vol 209 (3) ◽  
pp. 873-879 ◽  
Author(s):  
K Brocklehurst ◽  
S M Mushiri ◽  
G Patel ◽  
F Willenbrock
Keyword(s):  
Negative Charge ◽  
Cysteine Proteinase ◽  
Catalytic Site ◽  
Side Chain ◽  
Cysteine Proteinases ◽  
Thiol Groups ◽  
Active Centre ◽  
Ph Range ◽  
Thiolate Anions ◽  
Kinetics Of

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.

scholarly journals 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain

1976 ◽  
Vol 159 (2) ◽  
pp. 235-244 ◽  
Author(s):  
M Shipton ◽  
T Stuchbury ◽  
K Brocklehurst
Keyword(s):  
Aspartic Acid ◽  
Acid Catalysis ◽  
Ph Dependence ◽  
Side Chain ◽  
Carboxyl Groups ◽  
Active Centre ◽  
Catalytic Sites ◽  
Ph Range ◽  
Thiol Proteinases ◽  
Active Centres

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25°C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M-1·s-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M-1·s-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.

scholarly journals Factors effecting the thermostability of cysteine proteinases from Carica papaya

1993 ◽  
Vol 214 (1) ◽  
pp. 129-134 ◽  
Author(s):  
Ian G. SUMNER ◽  
Gillian W. HARRIS ◽  
Mark A. J. TAYLOR ◽  
Richard W. PICKERSGILL ◽  
A. Jane OWEN ◽  
...  
Keyword(s):  
Carica Papaya ◽  
Cysteine Proteinases

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 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

1981 ◽  
Vol 197 (3) ◽  
pp. 739-746 ◽  
Author(s):  
K Brocklehurst ◽  
B S Baines ◽  
J P Malthouse
Keyword(s):  
Cysteine Proteinase ◽  
Thiol Group ◽  
Interactive System ◽  
Active Centre ◽  
Ph Values ◽  
Hydrophobic Binding ◽  
Rapid Purification ◽  
Active Centres ◽  
Covalent Chromatography

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.

scholarly journals Multiple personalities of the RNA polymerase active centre

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1316-1320 ◽  
Author(s):  
Nikolay Zenkin
Keyword(s):  
Rna Synthesis ◽  
Catalytic Properties ◽  
Amino Acid Content ◽  
Annual Conference ◽  
Active Centre ◽  
Catalytic Activities ◽  
Trigger Loop ◽  
Living Organisms ◽  
Unique Ability ◽  
Active Centres

Transcription in all living organisms is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Our understanding of the functioning of the active centre of RNAPs has transformed recently with the finding that a conserved flexible domain near the active centre, the trigger loop (TL), participates directly in the catalysis of RNA synthesis and serves as a major determinant for fidelity of transcription. It also appears that the TL is involved in the unique ability of RNAPs to exchange catalytic activities of the active centre. In this phenomenon the TL is replaced by a transcription factor which changes the amino acid content and, as a result, the catalytic properties of the active centre. The existence of a number of transcription factors that act through substitution of the TL suggests that the RNAP has several different active centres to choose from in response to external or internal signals. A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https://www.youtube.com/watch?v=79Z7iXVEPo4

scholarly journals Extractable aluminium, iron and manganese in mineral soils: I Dependence of extractability on the pH of oxalate, pyrophosphate and EDTA extractants

1989 ◽  
Vol 61 (2) ◽  
pp. 73-78 ◽  
Author(s):  
Raina Niskanen
Keyword(s):  
Supporting Electrolyte ◽  
Pka Values ◽  
Fe And Mn ◽  
Mineral Soils ◽  
Ph Range ◽  
Iron And Manganese

Al, Fe and Mn in two mineral soils were extracted by 0.05 M and 0.02 M oxalate and pyrophosphate and 0.02 M EDTA solutions the pH of which was adjusted to values ranging from 1.7 to 11.0. The extractability of metals tended to decrease as the pH rose and as the deprotonation of extractant acid, expressed as pKa values, progressed. The reduction in extractability of metals by oxalate was rather steep at pH > 4, whereas the extractability by pyrophosphate remained moderate at a wider pH range. The extractability of metals by EDTA (pH 3.6—7.3) was lower than that by oxalate and pyrophosphate. Extractability was lower in the absence of the studied oxyacid anions and with 0.01 M KCI as the supporting electrolyte at a pH between 2 and 11 than in their presence.

scholarly journals 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

1985 ◽  
Vol 227 (2) ◽  
pp. 511-519 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Thiol Group ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Cysteine Proteinases ◽  
Acidic Media ◽  
Active Centre ◽  
Ph Values ◽  
Cathepsin H ◽  
Covalent Chromatography

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.

Characterization ofTrichomonas vaginalishaemolysis

Parasitology ◽  
1990 ◽  
Vol 101 (2) ◽  
pp. 171-175 ◽  
Author(s):  
D. C. Dailey ◽  
C. Te-Hung ◽  
J. F. Alderete
Keyword(s):  
Incubation Period ◽  
Trichomonas Vaginalis ◽  
Human Erythrocytes ◽  
Haemolytic Activity ◽  
Cysteine Proteinases ◽  
Temperature Dependent ◽  
Chloromethyl Ketone ◽  
Ph Range ◽  
Culture Supernatants ◽  
Dependent Mechanism

The haemolytic activity of liveTrichomonas vaginalisorganisms was investigated. Optimal haemolysis of human erythrocytes was observed at a parasite to erythrocyte ratio of 1:5 during a 2 h incubation period. No haemolytic activity was detected in concentrated culture supernatants after overnight growth of trichomonads or when parasites were separated from erythrocytes by a 3 μm filter, suggesting a contact-dependent mechanism for haemolysis. The haemolytic activity was temperature-dependent and maximal haemolysis occurred at 37 °C. Treatment of trichomonads with metronidazole reduced levels of haemolysis by > 50%. Maximal haemolysis occurred at the pH range of the vagina during trichomoniasis.N-μ-tosyl-L-lysyl-chloromethyl ketone and iodoacetamide, inhibitors of trichomonad cysteine proteinases, reduced the haemolytic activity of live parasites.

scholarly journals Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya)

1986 ◽  
Vol 237 (1) ◽  
pp. 105-110 ◽  
Author(s):  
R A McKee ◽  
S Adams ◽  
J A Matthews ◽  
C J Smith ◽  
H Smith
Keyword(s):  
Northern Blot ◽  
Untranslated Region ◽  
Carica Papaya ◽  
Cysteine Proteinase ◽  
Leaf Tissue ◽  
The Other ◽  
Cdna Clones ◽  
Cysteine Proteinases ◽  
Base Pairs ◽  
Hybridization Probe

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3′ untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.

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