The reaction of 2,4,6-trinitrobenzene sulfonic acid with pancreatic elastase

1970 ◽  
Vol 48 (11) ◽  
pp. 1249-1259 ◽  
Author(s):  
Leticia Rao ◽  
T. Hofmann
Keyword(s):  
Rate Constant ◽  
Sulfonic Acid ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Trinitrobenzene Sulfonic Acid ◽  
Amino Group ◽  
Tyrosine Residues ◽  
Inactivation Rate Constant ◽  
Lysine Residues ◽  
Ph Range

The reaction of elastase with trinitrobenzene sulfonic acid was investigated in the pH range 9–12. Elastase was found to be inactivated by 2,4,6-trinitrobenzene sulfonic acid. The pH dependence of the pseudo first-order inactivation rate constant showed a pK of 10.3 and gave a Hill plot coefficient of 1.15. Trinitrophenol did not inactivate the enzyme. These results indicate that the inactivation is due to the covalent reaction of trinitrobenzene sulfonic acid with a single group in the enzyme. This group is not the N-terminal since the loss of N-terminal valine was considerably slower than the loss of activity at pH 10.5. The inactivation of elastase with 2,4-dinitrofluorobenzene also showed no correlation with the loss of the N-terminal. When the enzyme was exhaustively treated and fully inactivated with trinitrobenzene sulfonic acid at pH 10.5, the N-terminal valine and two out of three lysine residues were trinitrophenylated. No evidence for the loss of histidine was found. One of the tyrosine residues may be trinitrophenylated as judged from the molar extinction of the trinitrophenylated protein, but it has not been possible to isolate a trinitrophenylated tyrosine-containing peptide. The results can be interpreted in one of two ways: (a) trinitrophenylation of a group with a pK of 10.3, not involved in the activity, inactivates because the introduction of the trinitrophenyl residue causes a denaturation of the enzyme; or (b) a group with a pK of 10.3 controls the active conformation of the enzyme. The results do not exclude the possibility that the N-terminal plays an important role in the activity of the enzyme. Below pH 10.5 the reactivity of the N-terminal is low, indicating that it is buried.At pH 9.0 only the ε-amino group of lysine in position 224 reacted with trinitrobenzene sulfonic acid and full activity was retained. The second-order rate constant for the trinitrophenylation of this group was 25 times higher than that of the ε-amino group of the α-N-benzoyllysine.

Oxidation of Nickel(II) and Manganese(II) by Peroxodisulfate in Aqueous Solution in the Presence of Molybdate. Crystal Structure of the (NH4)6[NiMo9O32].6H2O Product

1992 ◽  
Vol 45 (12) ◽  
pp. 1943 ◽  
Author(s):  
SJ Dunne ◽  
RC Burns ◽  
GA Lawrance
Keyword(s):  
Rate Constant ◽  
Linear Dependence ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
X Ray Diffraction ◽  
X Ray ◽  
First Order ◽  
Oxidant Concentration ◽  
Ph Range ◽  
Kinetics Of

Oxidation of Ni2+,aq, by S2O82- to nickel(IV) in the presence of molybdate ion, as in the analogous manganese system, involves the formation of the soluble heteropolymolybdate anion [MMogO32]2- (M = Ni, Mn ). The nickel(IV) product crystallized as (NH4)6 [NiMogO32].6H2O from the reaction mixture in the rhombohedra1 space group R3, a 15.922(1), c 12.406(1) � ; the structure was determined by X-ray diffraction methods, and refined to a residual of 0.025 for 1741 independent 'observed' reflections. The kinetics of the oxidation were examined at 80 C over the pH range 3.0-5.2; a linear dependence on [S2O82-] and a non-linear dependence on l/[H+] were observed. The influence of variation of the Ni/Mo ratio between 1:10 and 1:25 on the observed rate constant was very small at pH 4.5, a result supporting the view that the precursor exists as the known [NiMo6O24H6]4- or a close analogue in solution. The pH dependence of the observed rate constant at a fixed oxidant concentration (0.025 mol dm-3) fits dequately to the expression kobs = kH [H+]/(Ka+[H+]) where kH = 0.0013 dm3 mol-1 s-1 and Ka = 4-0x10-5. The first-order dependence on peroxodisulfate subsequently yields a second-order rate constant of 0.042 dm3 mol-1 s-1. Under analogous conditions, oxidation of manganese(II) occurs eightfold more slowly than oxidation of nickel(II), whereas oxidation of manganese(II) by peroxomonosulfuric acid is 16-fold faster than oxidation by peroxodisulfate under similar conditions.

Studies on Horseradish Peroxidase. VII. A Kinetic Study of the Oxidation of Iodide by Horseradish Peroxidase Compound II

1971 ◽  
Vol 49 (18) ◽  
pp. 3059-3063 ◽  
Author(s):  
R. Roman ◽  
H. B. Dunford ◽  
M. Evett
Keyword(s):  
Ionic Strength ◽  
Horseradish Peroxidase ◽  
Kinetic Study ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Acid Dissociation ◽  
Ph Range ◽  
Compound Ii ◽  
Kinetics Of

The kinetics of the oxidation of iodide ion by horseradish peroxidase compound II have been studied as a function of pH at 25° and ionic strength of 0.11. The logarithm of the second-order rate constant decreases linearly from 2.3 × 105 to 0.1 M−1 s−1 with increasing pH over the pH range 2.7 to 9.0. The pH dependence of the reaction is explained in terms of an acid dissociation outside the pH range of the study.

scholarly journals Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence

1991 ◽  
Vol 275 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Y K Li ◽  
J Boggaram ◽  
L D Byers
Keyword(s):  
Isotope Effect ◽  
Rate Constant ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Acidic Group ◽  
Irreversible Inhibitors ◽  
Effects Of Ph ◽  
Solvent Isotope ◽  
Bell Shaped Curve

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

scholarly journals The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 335-339 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Native Enzyme ◽  
Liver Protein ◽  
Thiol Groups ◽  
Protein Disulphide Isomerase ◽  
Order Rate ◽  
Reactive Groups

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Open-chain nitrogen compounds. Part XV. A kinetic study of the hydrolysis of 1-aryl-3-aryloxymethyl-3-methyltriazenes and related triazenes

1992 ◽  
Vol 70 (8) ◽  
pp. 2224-2233 ◽  
Author(s):  
Keith Vaughan ◽  
Donald L. Hooper ◽  
Marcus P. Merrin
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Buffer System ◽  
Open Chain ◽  
Nucleophilic Displacement ◽  
Ether Oxygen ◽  
Rate Of Hydrolysis ◽  
Electron Withdrawing Group ◽  
Ph Range ◽  
Kinetics Of

The kinetics of hydyrolysis of a series of 1-aryl-3-aryloxymethyl-3-methyltriazenes, Ar-N=N-NMe-CH2OAr′, was studied over the pH range 2–7.5. Reactions were followed by the change in UV absorbance spectra of the triazenes. The aryloxymethyltriazenes decompose more slowly at pH 7.5 than the hydroxymethyltriazenes, Ar-N=NMe-CH2OH; the hydrolysis is favoured by the presence of an electron-withdrawing group in Ar′. A mixed isopropanol/buffer system was developed in order to improve solubility of the aryloxymethyl triazenes. Lowering the pH caused an increase in the rate of hydrolysis and under strongly acidic conditions an electron-withdrawing group in Ar′ actually slows down the reaction. A Hammett plot of the pseudo-first-order rate constant, kobs, is curved, indicating that two or more mechanisms operate simultaneously and that the contribution of each mechanism is substituent-dependent. A plot of kobs vs. [buffer] is linear; the slope of the plot affords the rate constant, kb for the buffer-catalyzed reaction for each substituent. A Hammett plot of kb vs. σ is linear with ρ = +0.55, suggesting that the buffer-catalyzed reaction involves nucleophilic displacement of the phenoxy group by the buffer anion. Further analysis afforded the specific acid-catalyzed rate constants, [Formula: see text], for each substituent; this component of the reaction has a negative ρ, consistent with a mechanism involving protonation at the ether oxygen. The postulation that specific acid catalysis is a component of the reaction mechanism was confirmed by the observation of a solvent deuterium isotope effect, 2.28 > kH/kD > 1.60. Only the p-NO2 and p-CN phenyloxymethyltriazenes showed any spontaneous decomposition.

scholarly journals The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain

1989 ◽  
Vol 259 (2) ◽  
pp. 443-452 ◽  
Author(s):  
D Kowlessur ◽  
M O'Driscoll ◽  
C M Topham ◽  
W Templeton ◽  
E W Thomas ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Ligand Binding ◽  
Transition State ◽  
Rate Constant ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Catalytic Site ◽  
Binding Interactions ◽  
Electrostatic Fields ◽  
Transition State Geometry

1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed.

The mechanism of the nonenzymatic iodination of tyrosine by molecular iodine

1988 ◽  
Vol 66 (9) ◽  
pp. 967-978 ◽  
Author(s):  
H. Brian Dunford ◽  
Adejare J. Adeniran
Keyword(s):  
Ph Dependence ◽  
Acid Catalyst ◽  
Order Rate Constant ◽  
Molecular Iodine ◽  
Base Catalysis ◽  
Slow Step ◽  
Rate Law ◽  
General Acid ◽  
Phenolic Group ◽  
Ph Range

Over the pH range 7–10, at very low buffer concentration, the nonenzymatic iodination of tyrosine obeys the rate law[Formula: see text]where kapp is the measured second order rate constant based upon the total initial concentrations of molecular iodine and tyrosine and K2 (units M) is the equilibrium constant for [Formula: see text]. The value of k′ is 3.5 × 10−8 M∙s−1. There are three plausible mechanisms that fit the experimental data. One, the simplest, is a concerted process in which hypoiodous acid attacks tyrosine with its phenolic group unionized. The other two involve the formation of an iodinated quinoid reactive intermediate species in a rapid pre-equilibrium between unionized tyrosine and either hypoiodous acid or molecular iodine. The pre-equilibrium, if it occurs, favors the initial reactants. It is followed by a slow step in which the quinoid is converted to mono-iodinated tyrosine. Positive deviations from the rate law for pH dependence indicate that some specific acid catalysis (H3O+) is occurring in the pH range 5–7. In the presence of sufficient buffer, general acid–base catalysis is observed with acetic acid acting as a general acid catalyst in the vicinity of pH 5 and carbonate acting as a general base at pH ~ 9.5. The nonenzymatic iodination of tyrosine occurs more rapidly as the pH is increased, in marked contrast to the peroxidase-catalyzed iodination, which has its optimum at low pH.

The pH Dependence of the Ruthenium-Catalyzed Ferricyanide Oxidation of Cyclohexanol

1989 ◽  
Vol 42 (8) ◽  
pp. 1273 ◽  
Author(s):  
RW Kaziro ◽  
JK Beattie
Keyword(s):  
Aqueous Solutions ◽  
Rate Constant ◽  
Ph Dependence ◽  
Catalytic Cycle ◽  
Ph Range ◽  
Catalyst Lifetime ◽  
Reduced State

The oxidation of cyclohexanol to cyclohexanone by ferricyanide in alkaline aqueous solutions is catalysed by the addition of chlororuthenium compounds. In solutions of pH less than 11 the progress of the reaction is limited by the decomposition of the catalyst in its reduced state. The catalyst lifetime can be lengthened by an increase in the concentration of the ferricyanide oxidant. In solutions of pH 11.3-11.9 either of the oxidation or the reduction steps of the catalytic cycle can be made rate determining, by adjustment of the relative concentrations of cyclohexanol and ferricyanide . The decrease in rate with increase in pH is due to the pH dependence of the reaction of the oxidized catalyst. The rate constant decreases from 26 to 15 dm3 mol-1 s-l between pH 11.3 and 11.9. The rate constant for the ferricyanide oxidation of the reduced catalyst is pH-independent at (6 � 2)×102 dm3 mol-1 s-1 at 298 K, over the same pH range.

Kinetics and mechanism of the reaction of dimethyl acetylphosphonate with water. Expulsion of a phosphonate ester from a carbonyl hydrate

1978 ◽  
Vol 56 (13) ◽  
pp. 1792-1795 ◽  
Author(s):  
Ronald Kluger ◽  
David C. Pire ◽  
Jik Chin
Keyword(s):  
Rate Constant ◽  
Electronic Effect ◽  
Order Rate Constant ◽  
Ion Concentration ◽  
First Order ◽  
Cleavage Reactions ◽  
Ph Range ◽  
Rapid Hydrolysis ◽  
Hydrolysis Of ◽  
Mechanism Of The Reaction

Dimethyl acetylphosphonate (DAP) is rapidly cleaved in water to acetate and dimethylphosphonic acid. The half time for reaction at pH 7, 25 °C is estimated to be 3 s. The reaction is first order in hydroxide ion concentration and first order in DAP concentration. Rates of reaction were measured over the pH range 3.8 to 6.5 at 25 °C, 6.5 and 7.0 at 5 °C, 4.5 to 6.5 at 35 °C, and 4.5 to 6.0 at 45 °C. The average observed second-order rate constant at 25 °C is 2.4 × 106M−1 s−1. DAP is converted rapidly to a hydrated carbonyl adduct. The mechanism for the formation of the observed products is proposed to be analogous to cleavage reactions of other carbonyl hydrates, proceeding from a monoanion conjugate in this case. The estimated rate constant for the unimolecular cleavage of the carbonyl hydrate anion is 2 × 103 s−1. The rapid hydrolysis of DAP results from energetically favourable formation of a hydrate due to the electronic effect of the phosphonate diester. This effect also promoles ionization of the hydrate. The ionized hydrate readily expels the phosphonate diester to achieve the overall rapid hydrolysis.

scholarly journals The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

1976 ◽  
Vol 159 (3) ◽  
pp. 513-527 ◽  
Author(s):  
J M Armstrong ◽  
D R Trentham
Keyword(s):  
Rate Constant ◽  
Active Sites ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Rate Equations ◽  
Buffer System ◽  
Exponential Process ◽  
Order Rate ◽  
Rate Profile

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 × 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 × 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.

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