Horseradish Peroxidase. XVII. Reactions of Compounds I and II with p-Aminobenzoic Acid in Deuterium Oxide

1975 ◽  
Vol 53 (11) ◽  
pp. 1563-1569 ◽  
Author(s):  
C. D. Hubbard ◽  
H. B. Dunford ◽  
W. D. Hewson
Keyword(s):  
Horseradish Peroxidase ◽  
Dissociation Constants ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Enzyme Concentration ◽  
Acid Dissociation ◽  
Aminobenzoic Acid ◽  
Kinetic Isotope

The kinetics of reactions of horseradish peroxidase compounds I and II (HRP-I and HRP-II, respectively) with p-aminobenzoic acid have been studied in ordinary water and in deuterium oxide solution over a pH (pD) range 3–10, at 25° and at an ionic strength of 0.11. Under the conditions of the experiments the rate of reaction is first order both in substrate concentration and in enzyme concentration in both solvents. An analysis of the pH dependence of the second order rate constant in H2O confirms the presence of two acid dissociation groups on the enzyme with pKa' of 8.6 and ∼0 for HRP-II, whereas for HRP-I the data suggest a pKa of 5.1 on the enzyme and reveal, as previously shown, the influence of the ionization of the substituted ammonium group of the substrate. In deuterium oxide the pD profiles are similar to those in water but significant shifts for both kinetic and acid dissociation constants are observed for both compounds.The numerical values of the isotope effects taken together with previous results in general confirm that with labile substrates the group of pKa 8.6 in H2O on HRP-II is involved in general acid catalysis. p-Aminobenzoic acid is intermediate between labile and somewhat unreactive substrates and behaves similarly to the ferrocyanide ion in that both acid dissociation groups (pKa's 8.6 and ∼0) are influential in the catalysis of substrate oxidation by HRP-II. The kinetic isotope effect for the HRP-I reaction with p-aminobenzoic acid at high pH (pD) is consistent with a rate determining proton transfer but the group of pKa 5.1 in H2O remains unidentified.

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 Horseradish peroxidase. XXIX. Reactions in water and deuterium oxide: cyanide binding, compound I formation, and reactions of compounds I and II with ferrocyanide

1978 ◽  
Vol 56 (22) ◽  
pp. 2844-2852 ◽  
Author(s):  
H. Brian Dunford ◽  
W. Donald Hewson ◽  
Håkan Steiner
Keyword(s):  
Hydrogen Peroxide ◽  
Horseradish Peroxidase ◽  
Kinetic Isotope Effect ◽  
Deuterium Oxide ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Compound I ◽  
Kinetic Isotope ◽  
Cyanide Binding ◽  
Compound Ii

The kinetics of the reactions of hydrogen peroxide and cyanide with native horseradish peroxidase, as well as reactions of compounds I and II with ferrocyanide have been studied in ordinary water and in deuterium oxide at 25 °C and ionic strength 0.11 using a stopped-flow apparatus. Rate constants for all reactions were measured over a wide range of acidity in both solvents from which equilibrium and kinetic isotope effects were evaluated. Protonation of an ionizable group on the enzyme with a pKa value of 4.15 ± 0.05 in water inhibits the reactions with both hydrogen peroxide and cyanide. A significant kinetic isotope effect, kH/kD = 1.6 ± 0.1, was measured for compound I formation whereas no significant kinetic isotope effect was found for cyanide binding. On the basis of these findings, a partial mechanism for compound I formation is proposed in which the group of pKa 4.15 plays a crucial role. The pH dependencies of the ferrocyanide reaction in the pH interval 4.5–10.8 confirmed the role of an acid group with a pKa of 5.2 for compound I and for compound II a pKa of 8.6 and another with a value lower than that encompassed by the pH range of the study. Equilibrium isotope effects were found but no kinetic isotope effects for either the reaction of compound I or of compound II This suggests that there are no rate-limiting proton transfers in the reactions between ferrocyanide and compounds I and II of horseradish peroxidase. The only reducing substrates which exhibit positive kH/kD values possess a labile proton.

scholarly journals Deuterium Isotope Effects on Permeation and Gating of Proton Channels in Rat Alveolar Epithelium

1997 ◽  
Vol 109 (4) ◽  
pp. 415-434 ◽  
Author(s):  
Thomas E. DeCoursey ◽  
Vladimir V. Cherny
Keyword(s):  
Deuterium Oxide ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Channel Gating ◽  
Alveolar Epithelium ◽  
Ionic Species ◽  
Alveolar Epithelial Cells ◽  
Proton Channels ◽  
Deuterium Isotope Effects ◽  
Solvent Isotope

The voltage-activated H+ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D2O, for water, H2O, on both the conductance and the pH dependence of gating were explored. D+ was able to permeate proton channels, but with a conductance only about 50% that of H+. The conductance in D2O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D+ than H+), suggesting that D+ interacts specifically with the channel during permeation. Evidently the H+ or D+ current is not diffusion limited, and the H+ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H+ (or D+) and not OH− is the ionic species carrying current. The voltage dependence of H+ channel gating characteristically is sensitive to pHo and pHi and was regulated by pDo and pDi in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H+ current activation was about three times slower (τact was larger) in D2O than in H2O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H+ channel activation requires deprotonation of the channel. In contrast, deactivation (τtail) was slowed only by a factor ≤1.5 in D2O. The results are interpreted within the context of a model for the regulation of H+ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D2O can be explained if the pKa of the external regulatory site is ∼0.5 pH U higher in D2O.

scholarly journals Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A

1993 ◽  
Vol 289 (1) ◽  
pp. 185-193 ◽  
Author(s):  
W L Mock ◽  
D J Freeman ◽  
M Aksamawati
Keyword(s):  
Metal Ion ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Bound Water ◽  
Acid Dissociation ◽  
Lewis Acidity ◽  
Carboxypeptidase A ◽  
A Value ◽  
Straight Line

Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.

Secondary α-deuterium kinetic isotope effects for the E2 reaction of the 2-phenylethyl halides with tert-butoxide ion in tert-butyl alcohol

1985 ◽  
Vol 63 (1) ◽  
pp. 100-102 ◽  
Author(s):  
Peter James Smith ◽  
Kanchugarakoppal S. Rangappa ◽  
Kenneth Charles Westaway
Keyword(s):  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Order Rate Constant ◽  
Butyl Alcohol ◽  
Tert Butyl ◽  
Kinetic Isotope ◽  
Tert Butyl Alcohol ◽  
Elimination Reactions ◽  
Leaving Groups ◽  
High Base

Secondary α-deuterium kinetic isotope effects have been determined for the elimination reactions of 2-phenylethyl halides with tert-butoxide in tert-butyl alcohol at 40 °C in the presence and absence of the crown ether 18C6. The second-order rate constant k2 and the normal (kH/kD)α effect remained constant when the tert-butoxide concentration was varied for reaction of the iodo and bromo compounds. However, both the magnitude of k2 and the secondary α-deuterium isotope effect were significantly dependent on [t-BuO−] when chlorine and fluorine are the leaving groups. It is noteworthy that (kH/kD)α is inverse for the reaction of both the chloro and fluoro compounds at "low" base concentrations and normal at "high" base concentrations. These results are discussed in terms of both syn- and anti-elimination pathways promoted by various associated and dissociated base species. It is suggested that the (kH/kD)α effect may be useful as a criterion for determining the stereochemistry of E2 elimination reactions.

Studies on Horseradish Peroxidase. XI. On the Nature of Compounds I and II as Determined from the Kinetics of the Oxidation of Ferrocyanide

1973 ◽  
Vol 51 (4) ◽  
pp. 582-587 ◽  
Author(s):  
M. L. Cotton ◽  
H. B. Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Rate Constant ◽  
Active Sites ◽  
Ph Dependence ◽  
Order Rate Constant ◽  
Second Order ◽  
Compound I ◽  
Order Rate ◽  
Compound Ii ◽  
Kinetics Of

In order to investigate the nature of compounds I and II of horseradish peroxidase, the kinetics were studied of ferrocyanide oxidation catalyzed by these compounds which were prepared from three different oxidizing agents. The pH dependence of the apparent second-order rate constant for ferrocyanide oxidation by compound I, prepared from ethyl hydroperoxide and m-chloroperbenzoic acid, was interpreted in terms of an ionization on the enzyme with a pKa = 5.3, identical to that reported previously for hydrogen peroxide. The second-order rate constant for the compound II-ferrocyanide reaction also showed the same pH dependence for the three oxidizing substrates. However, with more accurate results, the compound II-ferrocyanide reaction was reinterpreted in terms of a single ionization with pKa = 8.5. The same dependence of ferrocyanide oxidation on pH suggests structurally identical active sites for compounds I and II prepared from the three different oxidizing substrates.

The pH dependence of xanthine oxidase catalysis in basic solution

1980 ◽  
Vol 58 (5) ◽  
pp. 394-398 ◽  
Author(s):  
John W. Bunting ◽  
Keith R. Laderoute ◽  
Donald J. Norris
Keyword(s):  
Xanthine Oxidase ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Kinetic Isotope Effects ◽  
Similar Rate ◽  
Basic Solution ◽  
Kinetic Isotope ◽  
Steady State Kinetics ◽  
Pyridinium Cations ◽  
Kinetics Of

The steady-state kinetics of the oxidation of the following six heteroaromatic substrates by xanthine oxidase have been investigated over the range pH 9.0–11.1 at 25 °C, ionic strength 0.1: 1-methylquinolinium, 6-methoxy-1-methylquinolinium, 1-methylnicotinamide, 3-acetyl-1-methylpyridinium, and 1-(4-methoxyphenyl)pyridinium cations and 1-methylnicotinate zwitterion. For the first four of these species, kc and Km were evaluated as a function of pH while only kc/Km was accessible in the latter two cases. Where available, kc is pH independent, whereas plots of log (kc/Km) vs. pH are linear with slopes in the range 0.54–1.17.The rates of enzymic oxidation of the 1-methylquinolinium cation and its 2-deuterio derivative were investigated and kinetic isotope effects were calculated at pH 9.8 and 10.6: kcH/kcD = 1.7 and KmH/KmD = 0.4 at each pH. Detailed comparisons of the oxidation of heteroaromatic cations and xanthine-derived substrates indicate that similar rate-determining steps control the enzymic oxidations of these two classes of substrate.

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