scholarly journals Evidence for association-activation effects in reactions of papain from studies on its reactivity towards isomeric two-protonic-state reactivity probes

1979 ◽  
Vol 183 (2) ◽  
pp. 369-373 ◽  
Author(s):  
K Brocklehurst ◽  
J A L Herbert ◽  
R Norris ◽  
H Suschitzky
Keyword(s):  
Kinetic Study ◽  
Thiol Group ◽  
Probe Molecule ◽  
Leaving Group ◽  
Rate Maximum ◽  
Neutral Media ◽  
Imidazolium Ion

4-(N-Aminoethyl 4-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole was synthesized and evaluted as a two-protonic-state reactivity probe by kinetic study of its reactions with papain (EC 3.4.22.2) and with benzimidazol-2-ylmethanethiol. Evidence is presented to suggest that: (i) both this probe molecule and its 2-pyridyl isomer bind to papain; (ii) the binding is followed by a change in the environment of the thiol group of cysteine-25; (iii) the striking rate maximum in neutral media observed in the reaction of papain with the 2-pyridyl isomer but not with the 4-pyridyl isomer arises from association of the 2-pyridyl leaving group with the imidazolium ion of histidine-159.

Kinetic Study of the Aminolysis and Pyridinolysis ofO-Phenyl andO-EthylO-(2,4-Dinitrophenyl) Thiocarbonates. A Remarkable Leaving Group Effect

2004 ◽  
Vol 69 (7) ◽  
pp. 2411-2416 ◽  
Author(s):  
Enrique A. Castro ◽  
María Cubillos ◽  
Margarita Aliaga ◽  
Sandra Evangelisti ◽  
José G. Santos
Keyword(s):  
Kinetic Study ◽  
Group Effect ◽  
Leaving Group

scholarly journals Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate

1982 ◽  
Vol 201 (1) ◽  
pp. 101-104 ◽  
Author(s):  
K Brocklehurst ◽  
S M Mushiri ◽  
G Patel ◽  
F Willenbrock
Keyword(s):  
Catalytic Triad ◽  
Interactive Systems ◽  
Alkaline Media ◽  
Active Centre ◽  
Wide Range ◽  
Catalytic Sites ◽  
Rate Maximum ◽  
Neutral Media ◽  
Acidic And Alkaline Media ◽  
Kinetics Of

1. The pH-dependences of the second-order rate constants (k) for the alkylation by chloroacetate of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) were determined over a wide range of pH at 25 degrees C at I 0.1. 2. The main feature of both pH-k profiles is a striking rate maximum at pH6 (characterizing parameters in both cases pKI approx. 3.5, pKII approx. 8.4 and pH-independent rate constant approximately kXH 2.5-3.0 M-1 . s-1). 3. The profile for the ficin reaction contains a plateau at high pH, with approximately kX 0.10 M-1 . s-1; if an analogous plateau exists in the papain reaction, approximately kX ix much lower, less than 0.02 M-1 . s-1. 4. Both enzymes appear to contain closely similar thiolate-imidazolium interactive systems at pH6, but differences in their behaviour in more-acidic media and in alkaline media suggest differences in interaction with the postulated carboxylate component of the putative catalytic triad.

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 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.

Kinetics of hydrolysis of n-hexyl bromide, n-heptyl bromide, and n-octyl bromide in alkaline and neutral media

1969 ◽  
Vol 22 (1) ◽  
pp. 137 ◽  
Author(s):  
B Singh ◽  
K Behari ◽  
B Krishna
Keyword(s):  
Kinetic Study ◽  
Rate Constants ◽  
Kinetics Of Hydrolysis ◽  
Different Temperatures ◽  
Neutral Media ◽  
Kinetics Of ◽  
Hydrolysis Of

Data obtained in the kinetic study of the hydrolysis of n-hexyl, n-heptyl, and n-octyl bromide in alkaline and neutral media at different temperatures and in varying ethanol-water mixtures are reported. The rate constants for the three reactions (bimolecular and unimolecular substitution, and bimolecular elimination) which occur simultaneously have been calculated for each compound.

Kinetic study of formation/dissociation of Cu(II) and Zn(II) complexes of cyclen macrocyclic ligand with pendant thiol group

Polyhedron ◽  
2013 ◽  
Vol 62 ◽  
pp. 268-273 ◽  
Author(s):  
Romana Ševčíková ◽  
Přemysl Lubal ◽  
Maria Paula Cabral Campello ◽  
Isabel Santos
Keyword(s):  
Kinetic Study ◽  
Macrocyclic Ligand ◽  
Thiol Group
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