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

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.

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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

Biochemical Journal ◽

10.1042/bj1930819 ◽

1981 ◽

Vol 193 (3) ◽

pp. 819-823 ◽

Cited By ~ 13

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.

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

Biochemical Journal ◽

10.1042/bj2960563 ◽

1993 ◽

Vol 296 (3) ◽

pp. 563-569 ◽

Cited By ~ 3

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.

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Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin

Biochemical Journal ◽

10.1042/bj1910707 ◽

1980 ◽

Vol 191 (3) ◽

pp. 707-718 ◽

Cited By ~ 8

Author(s):

K Brocklehurst ◽

J P G Malthouse

Keyword(s):

Aspartic Acid ◽

Catalytic Mechanism ◽

Striking Difference ◽

Compound I ◽

Active Centre ◽

Aspartic Acid Residue ◽

Wide Range ◽

Cationic Site ◽

Hydrophobic Binding ◽

Kinetics Of

The kinetics of the reactions of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) with the two-protonic-state reactivity probes 2,2′-dipyridyl disulphide, n-propyl 2-pyridyl disulphide and 4-(N-aminoethyl 2′-pyridyl disulphide)- 7-nitrobenzo-2-oxa-1,3-diazole (compound I) were studied over a wide range of pH. Differences between the reactivities of ficin and papain towards the cationic forms of the alkyl 2-pyridyl disulphide probes suggest that ficin contains a cationic site without exact analogue in papain, and the striking difference in the shapes of the pH-rate profiles for the reactions of the two enzymes with compound (1) suggests differences in the mobilities or dispositions of the active-centre histidine imidazole groups with respect to relevant hydrophobic binding areas. The evidence from reactivity-probe studies that the papain catalytic mechanism involves substantial repositioning of the active-centre imidazole group during the catalytic act does not apply also to ficin. If ficin contains an aspartic acid residue analogous to aspartic acid-158 in papain, the pKa of its carboxy group is probably significantly lower than the pKa of the analogous group in papain.

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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

Biochemical Journal ◽

10.1042/bj2090873 ◽

1983 ◽

Vol 209 (3) ◽

pp. 873-879 ◽

Cited By ~ 11

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.

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Hydrogen bonding in radical solution copolymerization kinetics of acrylates and methacrylates: a comparison of hydroxy- and methoxy-functionality

Polymer Chemistry ◽

10.1039/c7py00185a ◽

2017 ◽

Vol 8 (12) ◽

pp. 1943-1952 ◽

Cited By ~ 15

Author(s):

Jan E. S. Schier ◽

David Cohen-Sacal ◽

Robin A. Hutchinson

Keyword(s):

Experimental Data ◽

Hydrogen Bonding ◽

Growth Parameters ◽

Pulsed Laser ◽

Side Chain ◽

Chain Growth ◽

Kinetic Chain ◽

Kinetics Of ◽

Radical Solution ◽

Copolymerization Kinetics

Experimental data obtained via pulsed laser polymerization are used to distinguish the influence of H-bonding on kinetic chain-growth parameters from that of side-chain heteroatoms.

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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

Biochemical Journal ◽

10.1042/bj1710385 ◽

1978 ◽

Vol 171 (2) ◽

pp. 385-401 ◽

Cited By ~ 19

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.

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Synthesis and kinetic evaluation of 2-(N′-acetyl-l-phenylalanylamino)ethyl 2′-pyridyl disulphide as a two-protonic-state reactivity probe with S2-subsite specificity for papain: evidence that the alignment of the active-centre imidazolium ion of papain with the leaving group of a substrate depends on the interaction in the S2-subsite

Biochemical Society Transactions ◽

10.1042/bst0100216 ◽

1982 ◽

Vol 10 (4) ◽

pp. 216-217 ◽

Cited By ~ 3

Author(s):

GEETA PATEL ◽

KEITH BROCKLEHURST

Keyword(s):

Active Centre ◽

Kinetic Evaluation ◽

Leaving Group ◽

Imidazolium Ion ◽

S2 Subsite

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Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding

Biochemical Journal ◽

10.1042/bj2440173 ◽

1987 ◽

Vol 244 (1) ◽

pp. 173-181 ◽

Cited By ~ 18

Author(s):

K Brocklehurst ◽

D Kowlessur ◽

M O'Driscoll ◽

G Patel ◽

S Quenby ◽

...

Keyword(s):

Hydrogen Bonding ◽

Binding Site ◽

Ph Dependence ◽

Catalytic Site ◽

Amide Bond ◽

Side Chain ◽

Specific Substrate ◽

Cysteine Proteinases ◽

Pyridyl Nitrogen ◽

Compound Ii

1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2′-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2′-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2′-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Biochemical Journal ◽

10.1042/bj1670799 ◽

1977 ◽

Vol 167 (3) ◽

pp. 799-810 ◽

Cited By ~ 12

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.

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