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 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 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 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 Purification, Characterization, and Effect of Thiol Compounds on Activity of the Erwinia carotovora L-Asparaginase

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Suchita C. Warangkar ◽  
Chandrahas N. Khobragade
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Inverse Relationship ◽  
Reduced Glutathione ◽  
Erwinia Carotovora ◽  
Thiol Group ◽  
Thiol Groups ◽  
Thiol Compounds ◽  
Asparaginase Activity ◽  
And Control

L-asparaginase was extracted from Erwinia carotovora and purified by ammonium sulfate fractionation (60–70%), Sephadex G-100, CM cellulose, and DEAE sephadex chromatography. The apparent Mr of enzyme under nondenaturing and denaturing conditions was 150 kDa and  kDa, respectively. L-asparaginase activity was studied in presence of thiols, namely, L-cystine (Cys), L-methionine (Met), N-acetyl cysteine (NAC), and reduced glutathione (GSH). Kinetic parameters in presence of thiols (10–400 M) showed an increase in values (2000, 2223, 2380, 2500, and control 1666.7 moles ) and a decrease in values (0.086, 0.076, 0.062, 0.055 and control 0.098 mM) indicating nonessential mode of activation. values displayed propensity to bind thiols. A decrease in ratio in concentration plots showed inverse relationship between free thiol groups (NAC and GSH) and bound thiol group (Cys and Met). Enzyme activity was enhanced in presence of thiol protecting reagents like dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and GSH, but inhibited by p-chloromercurybenzoate (PCMB) and iodoacetamide (IA).

In vivo measurements of pulmonary angiotensin-converting enzyme kinetics. I. Theory and error analysis

1995 ◽  
Vol 78 (3) ◽  
pp. 1158-1168 ◽  
Author(s):  
J. Markham ◽  
T. J. McCarthy ◽  
M. J. Welch ◽  
D. P. Schuster
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzyme Kinetics ◽  
Kinetic Parameters ◽  
Converting Enzyme ◽  
Activity Data ◽  
Time Activity ◽  
Fast Kinetics ◽  
Trans Conformer ◽  
Kinetics Of

We developed a procedure for measuring pulmonary angiotensin-converting enzyme kinetics with fluorine-18 fluorocaptopril and positron emission tomography (PET). The method is based on the application of a compartmental receptor model that represents the kinetics of two species of ligand, presumably the trans and cis conformers of captopril. The input function was characterized and includes corrections for the labeled metabolites of fluorocaptopril. Application of the procedure to lung time-activity data obtained with PET produced estimates of kinetic parameters demonstrating fast kinetics for one conformer and slower kinetics for the other. Simulation studies were performed to evaluate the sensitivity of the estimated parameters to errors in the model assumptions and in measured values for variables required for analysis of the PET data. Estimates for two of the kinetic parameters, the amount of perfused unbound functional enzyme normalized to regional lung volume and the association rate constant for the trans conformer, were relatively stable even with large errors in the input data, varying < 30% from true values for all perturbations. Thus, the procedure produces reliable estimates of the kinetics of the trans conformer of captopril as well as theoretical curves that are close to the observed data.

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 Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups

1976 ◽  
Vol 157 (2) ◽  
pp. 479-482 ◽  
Author(s):  
T J Visser ◽  
I Does-Tobé ◽  
R Docter ◽  
G Hennemann
Keyword(s):  
Rat Liver ◽  
Essential Role ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Enzymic Activity ◽  
Marker Enzyme ◽  
Converting Enzyme ◽  
Thiol Groups ◽  
Liver Homogenates

Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.

scholarly journals The reactivity of thiol groups and the subunit structure of aldolase

1970 ◽  
Vol 117 (2) ◽  
pp. 291-298 ◽  
Author(s):  
P. J. Anderson ◽  
R. N. Perham
Keyword(s):  
Group Analysis ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Subunit Structure ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Prolonged Incubation ◽  
Nitrobenzoic Acid

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-14C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5′-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.

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.

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