scholarly journals Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system

1978 ◽  
Vol 171 (2) ◽  
pp. 385-401 ◽  
Author(s):  
M Shipton ◽  
K Brochlehurst
Keyword(s):  
Thiol Group ◽  
Interactive System ◽  
Pyridyl Ring ◽  
Thiol Groups ◽  
Sulphur Atom ◽  
Active Centre ◽  
Nucleophilic Reactivity ◽  
Imidazolium Ion ◽  
Active Centres

1.2,2′-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.

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 Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2′-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups

1982 ◽  
Vol 205 (1) ◽  
pp. 205-211 ◽  
Author(s):  
B S Baines ◽  
K Brocklehurst
Keyword(s):  
Carica Papaya ◽  
Cysteine Proteinase ◽  
Thiol Group ◽  
Catalytic Site ◽  
Interactive System ◽  
Ionization State ◽  
Active Centre ◽  
Ph Values ◽  
Activity Loss

1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely ‘essential’ for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2′-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.

scholarly journals Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols

1977 ◽  
Vol 167 (3) ◽  
pp. 799-810 ◽  
Author(s):  
M Shipton ◽  
K Brocklehurst
Keyword(s):  
High Electron ◽  
Thiol Groups ◽  
Active Centre ◽  
Enzyme Substrate ◽  
Mechanistic Basis ◽  
A Site ◽  
Kinetics Of ◽  
State Concentration ◽  
Imidazolium Ion ◽  
Active Centres

1. The characteristics of benzofuroxan (benzofurazan 1-oxide, benzo-2-oxa-1,3-diazole N-oxide) that relate to its application as a reactivity probe for the study of environments of thiol groups are discussed. 2. To establish a kinetic and mechanistic basis for its use as a probe, a kinetic study of its reaction with 2-mercaptoethanol was carried out. 3. This reaction appears to proceed by a rate-determining attack of the thiolate ion on one of the electrophilic centres of benzofuroxan (possibly C-6) to provide a low steady-state concentration of an intermediate adduct; rapid reaction of this adduct with a second molecule of thiol gives the disulphide and o-benzoquinone dioxime. 4. The effects of the different types of environment that proteins can provide on the kinetic characteristics of reactions of thiol groups with benzofuroxan are delineated. 5. Benzofuroxan was used as a thiolspecific reactivity probe to investigate the active centres of papain (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). The results support the concept that the active centres of all three enzymes either contain a nucleophilic thiolate ion whose formation is characterized by a pKa of 3-4 and whose reaction with an electrophile can be assisted by interaction of a site of high electron density in the electrophile with active-centre imidazolium ion of pKa 8-9, or can provide such ions by protonic redistribution in enzyme-reagent or enzyme-substrate complexes.

scholarly journals A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2'- and 4,4'- dipyridyl disulphides as reactivity probes

1980 ◽  
Vol 185 (1) ◽  
pp. 217-222 ◽  
Author(s):  
J P G Malthouse ◽  
K Brocklehurst
Keyword(s):  
Dielectric Constant ◽  
Physicochemical Properties ◽  
Solvent Effect ◽  
Rate Constants ◽  
Kinetic Method ◽  
Thiol Group ◽  
Analogous Reaction ◽  
Thiol Groups ◽  
Active Centre ◽  
Effect Of Solvent

1. Whereas the second-order rate constants for the reaction of the thiolate ion of 2-mercaptoethanol with 4,4'-dipyridyl disulphide (k4PDS) and with 5,5'-dithiobis-2-nitrobenzoate dianion increase with decreasing dielectric constant of the solvent, or remain unchanged, the rate constant for the analogous reaction with 2,2'-dipyridyl disulphide (k2PDS) decreases. This anomalous solvent effect and other unusual physicochemical properties of 2,2'-dipyridyl disulphide are discussed. 2. The differential effect of solvent on the reactions of thiolate ion with the 2,2'- and 4,4'-dipyridyl disulphides is shown to provide a method of characterizing solvent environments of thiol groups in proteins by a reactivity-probe method that should not suffer from the usual drawback associated with the existence of steric or binding effects of unknown magnitude. Application of the method to ficin (EC 3.4.22.3) suggests that its active-centre thiol group resides in a relatively hydrophobic environment. 3. The pH-k profile for the reaction of ficin with 4,4'-dipyridyl disulphide is reported.

scholarly journals Evidence that the lack of high catalytic activity of thiolsubtilisin towards specific substrates may be due to an inappropriately located proton-distribution system. Demonstration of highly nucleophilic character of the thiol group of thiolsubtilisin in the catalytically relevant ionization state of the active centre by use of a two-protonic-state reactivity probe

1981 ◽  
Vol 193 (3) ◽  
pp. 819-823 ◽  
Author(s):  
K Brocklehurst ◽  
J P G Malthouse
Keyword(s):  
Distribution System ◽  
Thiol Group ◽  
High Rate ◽  
Interactive System ◽  
High Catalytic Activity ◽  
Ionization State ◽  
Active Centre ◽  
Definitive Evidence ◽  
Rate Of Reaction ◽  
Kinetics Of

The active centre of the semi-synthetic enzyme thiolsubtilisin was investigated by studying the kinetics of the reaction of the thiol group of cysteine-221 with the thiol-specific two-protonic-state reactivity probe 2,2′-dipyridyl disulphide. The three-states criterion [Brocklehurst (1974) Tetrahedron 30, 2397-2407] was used to provide definitive evidence of the existence of a thiol–imidazole interactive system in acidic media in which the sulphur atom possesses highly nucleophilic character. The lack of catalytic competence of thiolsubtilisin despite its possession of the requisite nucleophilic capability is discussed. The exceedingly high rate of reaction of thiolsubtilisin with 2,2′-dipyridyl disulphide at pH 4–5 is shown to constitute a rapid and convenient active-site titration in which intact thiol–imidazole interaction is detected even in the presence of other thiols.

scholarly journals Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase

1993 ◽  
Vol 296 (3) ◽  
pp. 563-569 ◽  
Author(s):  
L A Dalton ◽  
J O McIntyre ◽  
S Fleischer
Keyword(s):  
Enzyme Kinetics ◽  
Kinetic Parameters ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Reduction Product ◽  
Thiol Groups ◽  
Active Centre ◽  
Fold Reduction ◽  
Kinetics Of

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1′-azobis-(NN′-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.

scholarly journals Evidence that binding to the S2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25–histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain

1979 ◽  
Vol 183 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Keith Brocklehurst ◽  
J. Paul G. Malthouse ◽  
Michael Shipton
Keyword(s):  
Hydrogen Bonding ◽  
Thiol Group ◽  
Side Chain ◽  
Striking Difference ◽  
Pyridyl Nitrogen ◽  
Active Centre ◽  
Specific Reactivity ◽  
Kinetics Of ◽  
Imidazolium Ion ◽  
S2 Subsite

A method is proposed by which site-specific reactivity probes that exhibit different reactivities in two ionization states can be used to detect association–activation phenomena that involve repositioning of acid/base groups in enzyme active centres. The pH-dependences of the apparent second-order rate constants (k) for the reactions of the thiol group of papain (EC 3.4.22.2) with a series of two-protonic-state reactivity probes are compared. The short-chain probes, 2,2′-dipyridyl disulphide and n-propyl 2-pyridyl disulphide, react at pH6 in adsorptive complexes and/or transition states with geometries that do not permit hydrogen-bonding of the pyridyl nitrogen atom with the active-centre imidazolium ion, as evidenced by the rate minima at pH6 and the rate maxima at pH4 provided by reagent protonation. Only when the probe molecule, e.g. 4-(N-aminoethyl 2′-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole [compound(III)], contains a long hydrophobic side chain is the reaction characterized by maximal rates at about pH6, as in the acylation step of the catalytic act (at pH6, kcompound III/k2,2′-dipyridyl disulphide ≃ 100). It is proposed that this striking difference in profile shape may result from binding of the hydrophobic side chain of compound (III) possibly in the S2-subsite of papain, which promotes a change in catalytic-site geometry involving repositioning of the imidazolium ion of histidine-159 and hydrogen-bonding with the N atom of the leaving group, as has been postulated to occur in the acylation step of substate hydrolysis.

scholarly journals Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group

1993 ◽  
Vol 290 (1) ◽  
pp. 75-83 ◽  
Author(s):  
G W Mellor ◽  
S K Sreedharan ◽  
D Kowlessur ◽  
E W Thomas ◽  
K Brocklehurst
Keyword(s):  
Ph Dependence ◽  
Thiol Group ◽  
Catalytic Site ◽  
Interactive System ◽  
Stopped Flow ◽  
Thiol Groups ◽  
Fast Phase ◽  
Catalytically Active ◽  
Ph Range ◽  
Calpain Ii

1. Four calpain II heterodimers (80 kDa/30 kDa, 80 kDa/29 kDa, 80 kDa/26 kDa and 80 kDa/18 kDa) were isolated from fresh porcine kidney by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose CL-6B and subsequently on Reactive Red 120/agarose followed by f.p.l.c. on a Q-Sepharose Hi-Load 16/10 column. 2. The major component (80 kDa/30 kDa) was used to provide the catalytically active calpain II 80 kDa/18 kDa heterodimer by treatment with CaCl2; titration with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) in the presence of monothioglycerol showed the preparation to have 1.0 +/- 0.05 catalytic sites per molecule of heterodimer. 3. The 80 kDa/30 kDa heterodimer was separated from monothioglycerol and other low-molecular-mass material by gel filtration on Sephadex G-25 without loss of catalytic activity towards sulphanilic acid/azocasein in the presence of added Ca2+. On storage overnight at a concentration of 3 microM in KCl at 4 degrees C in the absence of Ca2+ the activator-free preparation still produced fully active 80 kDa/18 kDa heterodimer on addition of Ca2+. 4. Activator-free 80 kDa/30 kDa heterodimer (in the absence of Ca2+) reacts relatively slowly with ethyl 2-pyridyl disulphide at pH 5.9; over 5000 s five thiol groups per molecule react, all at similar rates. In the presence of 8 mM CaCl2 under otherwise identical conditions (and also in the pH range 3.8-10.4) an initial faster phase of reaction corresponding to approx. one thiol group per molecule of heterodimer is generated, but it is not cleanly separated from the subsequent slower reactions on the stopped-flow trace. This fast phase of reaction does not occur when E64-inactivated calpain II is substituted for active 80 kDa/18 kDa heterodimer. 5. Greatly improved resolution of the fast phase of reaction involving the catalytic-site thiol group was achieved by using 2,2′-dipyridyl disulphide (2-Py-S-S-2-Py) instead of ethyl 2-pyridyl disulphide. 6. The pH-dependence of the second-order rate constant (k) for the reaction of the catalytically active activator-free 80 kDa/18 kDa calpain II heterodimer with 2-Py-S-S-2-Py was studied by stopped-flow spectral analysis in the pH range approx. 3-8 without interference from reactions of other thiol groups. 7. The form of the pH-k profile establishes for the first time the existence of an interactive catalytic site system [probably containing a (Cys)-S-/(His)-Im+H ion pair] analogous to those present in monomeric non-Ca(2+)-activated cysteine proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe

1976 ◽  
Vol 159 (2) ◽  
pp. 221-234 ◽  
Author(s):  
J P Malthouse ◽  
K Brocklehurst
Keyword(s):  
Aspartic Acid ◽  
Structural Difference ◽  
Thiol Group ◽  
High Reactivity ◽  
Carboxyl Group ◽  
Active Centre ◽  
Ph Values ◽  
Rate Of Reaction ◽  
Covalent Chromatography

1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.

scholarly journals Synthesis, Characterization of Chitosan-Aluminum Oxide Nanocomposite for Green Synthesis of Annulated Imidazopyrazol Thione Derivatives

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1160
Author(s):  
Abir S. Abdel-Naby ◽  
Sara Nabil ◽  
Sarah Aldulaijan ◽  
Ibtisam M. Ababutain ◽  
Azzah I. Alghamdi ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Aluminum Oxide ◽  
Reaction Medium ◽  
Thiol Group ◽  
Docking Studies ◽  
Significant Loss ◽  
Gram Negative Bacteria ◽  
Organic Catalyst ◽  
Group 1

Chitosan-aluminum oxide nanocomposite was synthesized, characterized, and used as a green heterogeneous catalyst to synthesize novel imidazopyrazolylthione derivatives. Nanocomposite polymeric material was characterized by EDS-SEM and XRD. The powerful catalytic activity, and its base character of the nanocomposite, was used to synthesize imidazopyrazolylthione (1) in a good yield compared to traditional cyclocondensation synthesis. Using the nanocomposite catalyst, substitution of the thiol group (1) afforded the corresponding thiourea (2) and the corresponding ester (3). The efficiency of the nanocomposite over the traditional base organic catalyst, Et3N and NaOH, makes it an effective, economic, and reproducible nontoxic catalyst. Moreover, the heterogeneous nanocomposite polymeric film was easily isolated from the reaction medium, and recycled up to four times, without a significant loss of its catalytic activity. The newly synthesized derivatives were screened as antibacterial agents and showed high potency. Molecular docking was also performed for a more in-depth investigation. The results of the docking studies have demonstrated that the docked compounds have strong interaction energies with both Gram-positive and Gram-negative bacteria.

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