scholarly journals Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate

1983 ◽  
Vol 213 (3) ◽  
pp. 635-642 ◽  
Author(s):  
P F Leadlay ◽  
J Q Fuller
Keyword(s):  
Isotope Effect ◽  
Isotopic Exchange ◽  
Specific Radioactivity ◽  
Product Formation ◽  
Propionibacterium Shermanii ◽  
Mechanistic Pathway ◽  
Catalysed Reaction ◽  
Malonyl Coa ◽  
Independent Estimate ◽  
Tritium Release

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.

scholarly journals Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water

1983 ◽  
Vol 213 (3) ◽  
pp. 643-650 ◽  
Author(s):  
J Q Fuller ◽  
P F Leadlay
Keyword(s):  
Proton Transfer ◽  
Isotopic Exchange ◽  
Tritiated Water ◽  
Specific Radioactivity ◽  
Proton Donor ◽  
Partial Reaction ◽  
Propionibacterium Shermanii ◽  
Catalysed Reaction ◽  
Donor And Acceptor

The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under ‘irreversible’ conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.

scholarly journals Compartmentation between glycolysis and gluconeogenesis in rat liver

1968 ◽  
Vol 110 (2) ◽  
pp. 303-312 ◽  
Author(s):  
C. J. Threlfall ◽  
D. F. Heath
Keyword(s):  
Kinetic Analysis ◽  
Intravenous Injection ◽  
Rat Liver ◽  
Isotopic Exchange ◽  
Direct Evidence ◽  
Specific Radioactivity ◽  
Glycolytic Pathway ◽  
Glucose Phosphorylation

1. The specific radioactivity–time relationships of glucose, glucose 6-phosphate, glycerol 1-phosphate and UDP-glucose were determined in rat liver after the intravenous injection of [U−14C]fructose, and a kinetic analysis was carried out. The glucose 6-phosphate pool was found to be compartmented into gluconeogenic and glycolytic components, and evidence was obtained that the triose phosphates were similarly compartmented. The glycolytic pathway was fed by glycogenolysis and glucose phosphorylation. There was no direct evidence that glycogenolysis fed only the glycolytic pathway, but this interpretation would make the liver resemble other organs in this respect. 2. UDP-glucose was not formed solely from gluconeogenic glucose 6-phosphate, as there was some dilution of label in the intervening glucose 1-phosphate pool, probably from glycogenolysis, though other pathways cannot be excluded. 3. The data cannot be explained by isotopic exchange.

Identifying the potential of pulsed LED irradiation in synthesis: copper-photocatalysed C–F functionalisation

2018 ◽  
Vol 54 (36) ◽  
pp. 4589-4592 ◽  
Author(s):  
Thomas P. Nicholls ◽  
Johnathon C. Robertson ◽  
Michael G. Gardiner ◽  
Alex C. Bissember
Keyword(s):  
Visible Light ◽  
Product Formation ◽  
Proof Of Concept ◽  
Concept Study ◽  
Catalysed Reaction ◽  
First Time ◽  
Led Irradiation

The results of a proof-of-concept study demonstrate for the first time that pulsed LED irradiation enhances the rate of product formation and the yield of a visible light-mediated photoredox-catalysed reaction.

Chloroform as an accelerator of propane oxidation

1963 ◽  
Vol 274 (1358) ◽  
pp. 413-426 ◽  
Keyword(s):  
Gas Phase ◽  
Maximum Rate ◽  
Isotopic Exchange ◽  
Pressure Change ◽  
Product Formation ◽  
Pressure Measurements ◽  
Chain Mechanism ◽  
Phase Oxidation ◽  
Gas Phase Oxidation ◽  
Simple Chain

Chloroform and the other chloromethanes, except carbon tetrachloride, accelerate the gas-phase oxidation of propane in the 'low-temperature' region. The relation of pressure change to reactant consumption and final product formation is not significantly modified in the catalyzed reaction, which can still be followed by pressure measurements. The value of the maximum rate in the presence of chloroform is given fairly closely by the expression (( ρ max .) [CHCL 3 ])/( ρ max .) 0 = 1 + constant x [CHCI 3 ]/[ R H]. The form of this suggests that, in the rate-determining steps, chloroform and paraffin are involved in analogous processes, and the key step is postulated to be R O 2 · + CHCI 3 → R OOH + CCl 3 · which re-inforces the reaction R O 2 · + R H → R OOH + R · in competing with those steps normally leading to degradation of R O 2 · radicals. Since little or no isotopic exchange occurs when CDCl 3 is used in place of CHCl 3 , the radical CCl 3 · does not regenerate chloroform, but initiates chains of the type CCl 3 ·→ ·CCl 2 · + Cl·, Cl· + R H → HCl + R · A slow consumption of chloroform (the oxidation of which is unimportant in the absence of propane) occurs, together with a slow build-up of hydrogen chloride. With certain approximations, a simple chain mechanism reproduces the experimental kinetic formula.

Isotope Effect Studies on Elimination Reactions. VIII. The Mechanism of the 1,3-Elimination Reaction of 3-Phenylpropyltrimethylammonium Iodide with Amide Ion in Liquid Ammonia

1972 ◽  
Vol 50 (14) ◽  
pp. 2332-2343 ◽  
Author(s):  
K. C. Westaway ◽  
A. N. Bourns
Keyword(s):  
Isotope Effect ◽  
Hydrogen Isotope ◽  
Liquid Ammonia ◽  
Isotopic Exchange ◽  
Quaternary Salt ◽  
Nitrogen Isotope ◽  
Deuterium Substitution ◽  
Order Of Magnitude ◽  
Hydrogen Isotope Effect ◽  
Cis And Trans

Reaction of 3-phenylpropyltrimethylammonium iodide with potassium amide in liquid ammonia at −55 °C was found to give concurrent 1,3-elimination forming phenylcyclopropane and 1,2-elimination forming 3-phenylpropene and cis- and trans-1-phenylpropene. Deuterium tracer studies on the salt labelled at C-3 established that neither a carbene nor an ylide are intermediates in the 1,3-elimination process. Isotopic exchange at C-3 was shown to accompany the reaction of the deuterated salt in ordinary ammonia, but it was not detected in the reaction of unlabelled salt in ammonia-d3. A nitrogen isotope effect, k14/k15, of 1.022 ± 0.001 was found for the 1,3-elimination, while the corresponding hydrogen isotope effect, estimated from the effect of isotopic substitution on the 1,3- and 1,2-elimination ratio, was shown to exceed 20. The hydrogen isotope effect for the disappearance of undeuterated and deuterated salts (elimination and exchange) was approximately 7.4. These observations, as well as the influence of deuterium substitution in both the quaternary salt and the solvent on the relative rates of 1,3- and 1,2-elimination, have been shown to be in accord with an Elcb mechanism in which the rate constants for the conversion of the intermediate 3-carbanion to 1,3-elimination product and for return to reactant by abstraction of a proton from ammonia are of the same order of magnitude.

scholarly journals SECONDARY DEUTERIUM ISOTOPE EFFECT IN CHLORINE ISOTOPIC EXCHANGE REACTIONS IN ACETONITRILE

1972 ◽  
Vol 1 (12) ◽  
pp. 1223-1224 ◽  
Author(s):  
Nobuo Tanaka ◽  
Aritsune Kaji ◽  
Jun-ichi Hayami
Keyword(s):  
Isotope Effect ◽  
Isotopic Exchange ◽  
Exchange Reactions ◽  
Deuterium Isotope Effect

scholarly journals A semi-preparative enzymic synthesis of malonyl-CoA from [14C]acetate and 14CO2: labelling in the 1, 2 or 3 position

1994 ◽  
Vol 300 (2) ◽  
pp. 355-358 ◽  
Author(s):  
G Roughan
Keyword(s):  
Specific Radioactivity ◽  
Enzymic Synthesis ◽  
Malonyl Coa ◽  
14Co2 Labelling

A semi-preparative enzymic synthesis of [1-14C]malonyl-CoA from [1-14C]acetate and bicarbonate, and of [3-14C]malonyl-CoA from Na2(14)CO3 and acetate, was achieved by using chloroplasts rapidly isolated from 7-8-day-old pea shoots. Around 70% of the [1-14C]acetate was converted into malonyl-CoA in 2-3 h, and the specific radioactivity of [3-14C]malonyl-CoA synthesized in the system was 25-30 Ci/mol. Reactions were monitored and labelled products were purified by h.p.l.c.

scholarly journals Some quantitative aspects of the labelling of proteins with 125I by the iodine monochloride method

1971 ◽  
Vol 121 (1) ◽  
pp. 139-143 ◽  
Author(s):  
M. Ceska ◽  
A. V. Sjödin ◽  
F. Grossmüller
Keyword(s):  
Mathematical Model ◽  
Theoretical Model ◽  
Exchange Reaction ◽  
Isotopic Exchange ◽  
Specific Radioactivity ◽  
Iodine Monochloride ◽  
Molar Ratios ◽  
Isotopic Exchange Reaction ◽  
Iodination Reaction ◽  
The Mathematical Model

The labelling of proteins by the iodine monochloride method was studied by using a mathematical model. The equations used were primarily derived from the mass law equation of the isotopic exchange reaction between [125I]iodide and iodine monochloride. For convenient application, all equations were programmed into a computing desk-top calculator. To support the validity of the theoretical model, a series of iodinations of insulin were performed under various labelling conditions. The results of these experiments compare well with the theoretically derived values. Deviations from the theoretical values occurring at molar ratios of [125I]iodide to iodine monochloride < 0.1 and > 4.0 are explained and suggestions made about how to prevent them. The mathematical model was used to simulate the isotopic exchange, and the iodination reaction under various conditions, to study (a) the influence of the amount of [125I]iodide on the amount of [125I]iodine monochloride formed, (b) the influence of the specific radioactivity of [125I]iodide on the amount of [125I]iodine monochloride formed, and (c) the influence of the specific radioactivity of [125I]iodide on the number of millicuries needed for labelling to a desired extent.

Hydride transfer reactions. Oxidation of N-methylacridan by π acceptors

1977 ◽  
Vol 55 (14) ◽  
pp. 2741-2751 ◽  
Author(s):  
Allan K. Colter ◽  
Gunzi Saito ◽  
Frances J. Sharom
Keyword(s):  
Charge Transfer ◽  
Isotope Effect ◽  
Isotopic Exchange ◽  
Hydride Transfer ◽  
Transfer Reactions ◽  
Kinetic Measurements ◽  
Rate Of Reaction ◽  
Hydride Transfer Reactions ◽  
Isotope Partitioning

The oxidation of N-methylacridan (1) to N-methylacridinium ion (2) by the π acceptors p-benzoquinone (BQ), 7,7,8,8-tetracyanoquinodimethane (TCNQ), p-chloranil (CA), tetracyanoethylene (TCNE), and 2,3-dicyano-1,4-benzoquinone (DCBQ) has been investigated. Second-order rate constants in acetonitrile (AN) vary by a factor of more than 107 for this series. The rate of reaction of 1 with BQ increases 45-fold as the solvent composition is varied from AN to 50% (v/v) AN–water, but is insensitive to changes in buffer ratio or concentration in 75% AN. Spectroscopic evidence for charge-transfer complexing between the reactants was obtained with BQ and CA, and kinetic evidence for complexing was obtained with BQ in 75% AN. The primary isotope effect, p, calculated from the rates of oxidation of 1, 1-9-d, and 1-9,9-d2 (1(HH), 1(HD), and 1(DD)) in AN varied from 4.5 (TCNE) to 13 (BQ), while the secondary isotope effect, s, was approximately constant (∼1.1). Values of the isotope partitioning ratio, ipr (the ratio of 2(D) to 2(H) formed in reaction of 1(HD)) were determined for BQ in three AN-water mixtures and for CA, TCNE, and DCBQ in AN. For all systems studied, except BQ in 90% AN, where determination of the ipr is complicated by isotopic exchange between unreacted 1 and product (2), the ipr agrees with p/s from the kinetic measurements. These results are discussed in terms of mechanism and compared with those of other hydride transfer reactions involving dihydronicotinamide donors.

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