scholarly journals Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles

1998 ◽  
Vol 330 (1) ◽  
pp. 267-275 ◽  
Author(s):  
Ter-Mei HUANG ◽  
Hui-Chih HUNG ◽  
Tsu-Chung CHANG ◽  
Gu-Gang CHANG
Keyword(s):  
Aqueous Solution ◽  
Isotope Effect ◽  
Reverse Micelles ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Solvent System ◽  
Placental Alkaline Phosphatase ◽  
K Value ◽  
Kinetic Isotope ◽  
Human Placental Alkaline Phosphatase

Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2,4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were observed in reverse micelles. However, only one pKa value of 9.78-10.27 in aqueous solution was detected. Profiles of log V/K versus pL were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; at pL 10.4, the log V/K value was identical in water and deuterium. The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (ϕ) for deuterium for the transition state (ϕT) increased when the pH of the solution was raised. At pL 11.0, the ϕT was 1.07 in reverse micelles, which corresponds to the inverse-isotope effect of the reaction in this solvent system. Normal viscosity effects on kcat and kcat/Km were observed in aqueous solution, corresponding to a diffusional controlled physical step as the rate-limiting step. We propose that the rate-limiting step of the hydrolytic reaction changes from phosphate releasing in aqueous solution to a covalent phosphorylation or dephosphorylation step in reverse micelles.

Secondary Isotope Effects on the Ionization of 2-Nitropropane

1974 ◽  
Vol 52 (10) ◽  
pp. 1889-1896 ◽  
Author(s):  
A. J. Kresge ◽  
D. A. Drake ◽  
Y. Chiang
Keyword(s):  
Aqueous Solution ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Acid Dissociation ◽  
Methyl Groups ◽  
Anomalous Effect ◽  
Hydroxide Ion ◽  
Kinetic Isotope

The equilibrium isotope effect on the acid dissociation of 2-nitropropane in wholly aqueous solution at 25° was found to be KH/KD = 1.23 ± 0.03 for complete deuteration of both methyl groups; the kinetic isotope effect for reaction of the same substrate with hydroxide ion, kH/kD = 1.09 ± 0.01; and the kinetic isotope effect for reaction with tris-(hydroxymethyl)-methylamine, kH/kD = 1.10 ± 0.01; both of the latter also refer to wholly aqueous solution at 25° and are for complete deuteration of both methyl groups. It is shown that the equilibrium isotope effect is largely, and the kinetic isotope effects probably partly, hyper conjugative in origin, thus supporting a hyper conjugative explanation of the anomalous effect of methyl groups on nitroalkane ionization.

scholarly journals A kinetic-isotope-effect study of catalysis by Vibrio cholerae neuraminidase

1993 ◽  
Vol 294 (3) ◽  
pp. 653-656 ◽  
Author(s):  
X Guo ◽  
M L Sinnott
Keyword(s):  
Vibrio Cholerae ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Effect Study ◽  
K Value ◽  
Rate Determining Step ◽  
Kinetic Isotope ◽  
Leaving Group ◽  
Optimum Ph

Michaelis-Menten parameters for hydrolysis of seven aryl N-acetyl alpha-D-neuraminides by Vibrio cholerae neuraminidase at pH 5.0 correlate well with the leaving-group pKa (delta pK 3.0; beta 1g (V/K) = -0.73, r = -0.93; beta 1g (V) = -0.25; r = -0.95). The beta-deuterium kinetic-isotope effect, beta D2(V), for the p-nitrophenyl glycoside is the same at the optimum pH of 5.0 (1.059 +/- 0.010) as at pH 8.0 (1.053 +/- 0.010), suggesting that isotope effects are fully expressed with this substrate at the optimum pH. For this substrate at pH 5.0, leaving group 18O effects are 18(V) = 1.040 +/- 0.016 and 18(V/K) = 1.046 +/- 0.015, and individual secondary deuterium effects are beta proRD(V) = 1.037 +/- 0.014, beta proSD(V) = 1.018 +/- 0.015, beta proRD(V/K) = 1.030 +/- 0.017, beta proSD(V/K) = 1.030 +/- 0.017. All isotope effects, and the beta 1g(V/K) value are in accord with the first chemical step being both the first irreversible and the rate-determining step in enzyme turnover, with a transition state in which there is little proton donation to the leaving group, the C-O bond is largely cleaved, there is significant nucleophilic participation, and the sugar ring is in a conformation derived from the ground-state 2C5 chair. The apparent conflict between the beta 1g (V) value of -0.25 with all the kinetic-isotope-effect data can be resolved by the postulation of an interaction between the pi system of the aglycone ring and an anionic or nucleophilic group on the enzyme.

SOME DEUTERIUM KINETIC ISOTOPE EFFECTS: IV. β-DEUTERIUM EFFECTS IN WATER SOLVOLYSIS OF ETHYL, ISOPROPYL, AND tert-BUTYL COMPOUNDS

1960 ◽  
Vol 38 (11) ◽  
pp. 2171-2177 ◽  
Author(s):  
K. T. Leffek ◽  
J. A. Llewellyn ◽  
R. E. Robertson
Keyword(s):  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Alkyl Halides ◽  
Tert Butyl ◽  
Kinetic Isotope ◽  
Deuterium Isotope Effects ◽  
Intramolecular Forces

The secondary β-deuterium isotope effects have been measured in the water solvolytic reaction of alkyl halides and sulphonates for primary, secondary, and tertiary species. In every case the kinetic isotope effect was greater than unity (kH/kD > 1). This isotope effect may be associated with varying degrees of hyperconjugation or altered non-bonding intramolecular forces. The experiments make it difficult to decide which effect is most important.

Solvolysis of specifically deuterated 7-chloro- and 7-bromo-exo-2-norbornyl brosylates; unusually large γ-kinetic isotope effects in the absence of σ-participation

1980 ◽  
Vol 58 (16) ◽  
pp. 1738-1750 ◽  
Author(s):  
Nick Henry Werstiuk ◽  
George Timmins ◽  
Frank Peter Cappelli
Keyword(s):  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope

A series of specifically deuterated syn-7-chloro-, anti-7-chloro-, syn-7-bromo-, and anti-7-bromo-exo-2-norbornyl brosylates have been prepared and solvolyzed in NaOAc-buffered 80:20 EtOH–H2O. For solvolysis at 25 °C the γ-kinetic isotope effects (KIE's) for syn-7-chloro-exo-2-norbornyl brosylate-endo-6-d (1e), anti-7-chloro-exo-2-norbornyl brosylate-endo-6-d (2c), syn-7-bromo-exo-2-norbornyl brosylate-endo-6-d (1f), anti-7-bromo-exo-2-norbornyl brosylate-endo-6-d (2d), syn-7-chloro-exo-2-norbornyl brosylate-exo,exo-5,6-d2 (1g), anti-7-chloro-exo-2-norbornyl brosylate-exo,exo-5,6-d2 (2e) are 1.125 ± 0.007, 1.128 ± 0.005, 1.063 ± 0.008, 1.149 ± 0.020, 1.119 ± 0.011, and 1.115 ± 0.013, respectively. There is no detectable γ-kinetic isotope effect for solvolysis of anti-7-chloro-endo-2-norbornyl brosylate-endo-6-d(3a) and the β-KIE for anti-7-chloro-exo-2-norbornyl brosylate-exo-3-d(4a) is 1.111 ± 0.011. From a consideration of the possible sources of the unusually large secondary KIE's, we conclude that the exo-6-d and endo-6-d γ-KIE's likely are derived from a combination of effects rather than from participation of the C1—C6 bond in the ionization step.

Ketonization equilibria of phenol in aqueous solution

1999 ◽  
Vol 77 (5-6) ◽  
pp. 605-613 ◽  
Author(s):  
Marco Capponi ◽  
Ivo G Gut ◽  
Bruno Hellrung ◽  
Gaby Persy ◽  
Jakob Wirz
Keyword(s):  
Aqueous Solution ◽  
Proton Transfer ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Flash Photolysis ◽  
Equilibrium Constants ◽  
Thermodynamic Cycle ◽  
Acidity Constant ◽  
Kinetic Isotope ◽  
Carbon Acids

The two keto tautomers of phenol (1), cyclohexa-2,4-dienone (2) and cyclohexa-2,5-dienone (3), were generated by flash photolysis of appropriate precursors in aqueous solution, and the pH-rate profiles of their enolization reactions, 2 –> 1 and 3 –> 1, were measured. The rates of the reverse reactions, 1 –> 2 and 1 –> 3, were determined from the rates of acid-catalyzed hydron exchange at the ortho- and para-positions of 1; the magnitude of the kinetic isotope effect was assessed by comparing the rates of hydrogenation of phenol-2t and -2d. The ratios of the enolization and ketonization rate constants provide the equilibrium constants of enolization, pKE(2, aq, 25°C) = -12.73 ± 0.12 and pKE(3, aq, 25°C) = -10.98 ± 0.15. Combination with the acidity constant of phenol also defines the acidity constants of 2 and 3 through a thermodynamic cycle. These ketones are remarkably strong carbon acids: pKa(2) = -2.89 ± 0.12 and pKa(3) = -1.14 ± 0.15. They disappear by proton transfer to the solvent with lifetimes, τ(2) = 260 μs and τ(3) = 13 ms, that are insensitive to pH in the range from 3-10.Key words: proton transfer, tautomers, flash photolysis, kinetic isotope effect, pH-rate profiles.

scholarly journals Insights Into the Known 13C Depletion of Methane—Contribution of the Kinetic Isotope Effects on the Serine Hydroxymethyltransferase Reaction

2022 ◽  
Vol 9 ◽  
Author(s):  
Gerd Gleixner
Keyword(s):  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Serine Hydroxymethyltransferase ◽  
Chemical Degradation ◽  
Methyl Groups ◽  
Kinetic Isotope ◽  
Isotopic Pattern ◽  
Before And After ◽  
Metal Organic

We determined the kinetic isotope effect on the serine hydroxymethyltransferase reaction (SHMT), which provides important C1 metabolites that are essential for the biosynthesis of DNA bases, O-methyl groups of lignin and methane. An isotope effect on the SHMT reaction was suggested being responsible for the well-known isotopic depletion of methane. Using the cytosolic SHMT from pig liver, we measured the natural carbon isotope ratios of both atoms involved in the bond splitting by chemical degradation of the remaining serine before and after partial turnover. The kinetic isotope effect 13(VMax/Km) was 0.994 0.006 and 0.995 0.007 on position C-3 and C-2, respectively. The results indicated that the SHMT reaction does not contribute to the 13C depletion observed for methyl groups in natural products and methane. However, from the isotopic pattern of caffeine, isotope effects on the methionine synthetase reaction and on reactions forming Grignard compounds, the involved formation and fission of metal organic bonds are likely responsible for the observed general depletion of “activated” methyl groups. As metal organic bond formations in methyl transferases are also rate limiting in the formation of methane, they may likely be the origin of the known 13C depletion in methane.

ISOTOPE EFFECTS IN MIXTURES OF LIQUID H2O AND T2O

1966 ◽  
Vol 44 (6) ◽  
pp. 689-694 ◽  
Author(s):  
Mark Salomon
Keyword(s):  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Hydroxide Ion ◽  
Kinetic Isotope ◽  
Hydronium Ion ◽  
Mercury Cathode

Calculations are presented for the equilibrium tritium isotope effect involving water, hydronium ion, and hydroxide ion. The results are used to predict the kinetic isotope effect in the transfer of protons to a mercury cathode.

Kinetic evidence for the importance of solvent-separated ion pairs during the solvolyses of adamantylideneadamantyl derivatives

2004 ◽  
Vol 82 (9) ◽  
pp. 1336-1340
Author(s):  
Xicai Huang ◽  
Andrew J Bennet
Keyword(s):  
Ionic Strength ◽  
Transition State ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Ion Pairs ◽  
Kinetic Isotope Effects ◽  
Ion Pair ◽  
Salt Effects ◽  
Kinetic Isotope

The aqueous ethanolysis reactions of adamantylideneadamantyl tosylate, -bromide, and -iodide (1-OTs, 1-Br and 1-I) were monitored as a function of ionic strength. Special salt effects are observed during the solvolyses of both homoallylic halides, but not in the case of the tosylate 1-OTs. The measured α-secondary deuterium kinetic isotope effects for the solvolysis of 1-Br in 80:20 and 60:40 v/v ethanol–water mixtures at 25 °C are 1.110 ± 0.018 and 1.146 ± 0.009, respectively. The above results are consistent with the homoallylic halides reacting via a virtual transition state in which both formation and dissociation of a solvent-separated ion pair are partially rate-determining. While the corresponding transition state for adamantylideneadamantyl tosylate involves formation of the solvent-separated ion pair.Key words: salt effects, kinetic isotope effect, internal return, solvolysis, ion pairs.

scholarly journals Kinetics and Mechanism of the Decarboxylation of Pyrrole-2-carboxylic Acid in Aqueous Solution

1971 ◽  
Vol 49 (7) ◽  
pp. 1032-1035 ◽  
Author(s):  
G. E. Dunn ◽  
Gordon K. J . Lee
Keyword(s):  
Aqueous Solution ◽  
Ionic Strength ◽  
Carboxylic Acid ◽  
Isotope Effect ◽  
Anthranilic Acid ◽  
Kinetic Isotope Effect ◽  
Pyrrole Ring ◽  
First Order ◽  
Kinetic Isotope ◽  
Ph Range

The decarboxylation of pyrrole-2-carboxylic acid in aqueous buffers at 50° and ionic strength 1.0 has been found to be first order with respect to substrate at a fixed pH. As the pH is decreased, the rate constant increases slightly in the pH range 3–1, then rises rapidly from pH 1 to 10 M HCl. The 13C-carboxyl kinetic isotope effect is 2.8% in 4 M HClO4 and negligible at pH ~ 3. These observations can be accounted for by a mechanism, previously proposed for the decarboxylation of anthranilic acid, in which the species undergoing decarboxylation is the carboxylate ion protonated at the 2-position of the pyrrole ring. This intermediate can be formed both by ring-protonation of the carboxylate anion and by ionization of the ring-protonated acid. At low acidities ring-protonation is rate determining, but at higher acidities the rate of protonation exceeds that of decarboxylation.

scholarly journals Kinetic isotope effects for fast deuterium and proton exchange rates

2016 ◽  
Vol 18 (15) ◽  
pp. 10144-10151 ◽  
Author(s):  
Estel Canet ◽  
Daniele Mammoli ◽  
Pavel Kadeřávek ◽  
Philippe Pelupessy ◽  
Geoffrey Bodenhausen
Keyword(s):  
Exchange Rates ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Proton Exchange ◽  
Kinetic Isotope ◽  
Spin Pairs

By monitoring the effect of deuterium decoupling on the decay of transverse 15N magnetization in D–15N spin pairs during multiple-refocusing echo sequences, we have determined fast D–D exchange rates kD and compared them with fast H–H exchange rates kH in tryptophan to determine the kinetic isotope effect as a function of pH and temperature.

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