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 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.

Intermolecular proton transfer in catalysis by carbonic anhydrase V

1999 ◽  
Vol 77 (5-6) ◽  
pp. 726-732 ◽  
Author(s):  
J Nicole Earnhardt ◽  
Chingkuang Tu ◽  
David N Silverman
Keyword(s):  
Carbonic Anhydrase ◽  
Proton Transfer ◽  
Active Site ◽  
Rate Constants ◽  
Intramolecular Proton Transfer ◽  
Proton Donor ◽  
Marcus Theory ◽  
Intermolecular Proton Transfer ◽  
Donor And Acceptor ◽  
Active Site Cavity

The dehydration of bicarbonate catalyzed by carbonic anhydrase is accompanied by the transfer of a proton from solution to the zinc-bound hydroxide. We have investigated the properties of proton transfer from donors in solution, mostly derivatives of imidazole and pyridine, to a truncated mutant of carbonic anhydrase V with replacements that render the active site cavity less sterically constrained, Tyr 64 →> Ala and Phe 65 →> Ala. Catalysis was measured by determining the rate of exchange of 18O between the CO2-HCO3- system and water, and rate constants for proton transfer were estimated as the rate-limiting step in the release of H218O from the enzyme to solution. Each proton donor enhanced catalytic activity in a saturable manner. The resulting rate constants for proton transfer when compared with the values of pKa of the donor and acceptor gave a Brønsted plot of high curvature. These data could also be described by Marcus theory which showed an intrinsic barrier for intermolecular proton transfer near 0.8 kcal/mol and a work term or thermodynamic contribution to the free energy of reaction near 10 kcal/mol. This low intrinsic kinetic barrier for proton transfer is very similar to nonenzymic bimolecular proton transfer between nitrogen and oxygen acids and bases in solution. However, the significant thermodynamic contribution suggests appreciable involvement of solvent and active-site organization prior to proton transfer. These Marcus parameters are very similar to those describing intramolecular proton transfer from His 64 in carbonic anhydrase, suggesting similarities in the intra- and intermolecular proton transfer processes.Key words: carbonic anhydrase, proton transfer, Marcus theory, carbon dioxide.

A Potentiometric Study on Proton-Transfer Equilibria and Cationic Conjugation in Pyridine N-Oxide Systems in Acetone and Methanol

1996 ◽  
Vol 49 (9) ◽  
pp. 931 ◽  
Author(s):  
L Chmurzynski ◽  
E Kaczmarczyk ◽  
M Nesterowicz ◽  
G Wawrzyniak ◽  
Z Warnke
Keyword(s):  
Proton Transfer ◽  
Equilibrium Constants ◽  
Proton Donor ◽  
Heterocyclic Amines ◽  
Transfer Reactions ◽  
Oxide Systems ◽  
Experimental Systems ◽  
Proton Transfer Reactions ◽  
Potentiometric Titration Method

The potentiometric titration method has been used to study the equilibria of cationic in sytems formed by substituted pyridine N-oxides in the polar, non-aqueous solvents acetone and methanol. For comparison, the systems with trimethylamine N-oxide as a representative of aliphatic amine N-oxides and pyridine representing parent heterocyclic amines were also studied. The cationic heteroconjugation constants, i.e. the equilibrium constants for conjugation reactions between free and protonated N-bases leading to the formation of unsymmetric BHB'+ cations, were determined in experimental systems with and without proton transfer. It was found that there were significant differences in the values of the cationic heteroconjugation constants determined in these two acid-base systems. The proton-transfer reactions limit and even preclude the determination of the cationic heteroconjugation constants. On this basis it was concluded that the heteroconjugation constants should be determined in systems without proton transfer. In such systems, in the amphiprotic solvent methanol, cationic heteroconjugation was ascertained in all substituted pyridine N-oxide systems, the values of heteroconjugation constants being relatively low (logarithms of their values of the order of 2-2.5), and only negligible in systems involving trimethylamine N-oxide. A more pronounced tendency towards cationic heteroconjugation of the [OHO]+ type was observed in the aprotic protophobic acetone, where heteroconjugation constants were determined for all amine N-oxide systems studied including those containing protonated trimethylamine N-oxide as a proton donor. However, the values of the cationic heteroconjugation constants were found to be, in methanol likewise, relatively low (log KBHB'+ of the order of 2-3). On the contrary, a greater extent of cationic heteroconjugation equilibria was observed in methanol than in acetone in the case of systems containing pyridine, i.e. [NHO]+ type bridges formed by amine N-oxides and heterocyclic amines. In methanol the heteroconjugation constants turned out to be determinable for all such systems studied (logarithms of the equilibrium constants being of the same order as for N-oxide systems), whereas in acetone the hetero constants were indeterminable for all systems.

Capture and Isotopic Exchange Method for Water and Hydrogen Isotopes on Zeolite Catalysts up to Technical Scale for Pre-Study of Processing Highly Tritiated Water

2015 ◽  
Vol 67 (3) ◽  
pp. 483-486 ◽  
Author(s):  
Robert Michling ◽  
Adalbert Braun ◽  
Ion Cristescu ◽  
Helmut Dittrich ◽  
Manfred Glugla ◽  
...  
Keyword(s):  
Isotopic Exchange ◽  
Tritiated Water ◽  
Hydrogen Isotopes ◽  
Zeolite Catalysts ◽  
Exchange Method

scholarly journals A Solvent-Mediated Excited-State Intermolecular Proton Transfer Fluorescent Probe for Fe3+ Sensing and Cell Imaging

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 516
Author(s):  
You Qian ◽  
Fuchun Gong ◽  
Jiguang Li ◽  
Pan Ma ◽  
Hanming Zhu ◽  
...  
Keyword(s):  
Excited State ◽  
Proton Transfer ◽  
Fluorescent Probe ◽  
Cell Imaging ◽  
Cyano Group ◽  
Proton Donor ◽  
Cell Permeability ◽  
One Pot ◽  
Intermolecular Proton Transfer ◽  
Synthesis Strategy

Constructing excited-state intermolecular proton transfer (ESIPT-e) fluorophores represents significant challenges due to the harsh requirement of bearing a proton donor-acceptor (D-A) system and their matching proton donating-accepting ability in the same molecule. Herein, we synthesized a new-type ESIPT-e fluorophor (2-APC) using the “four-component one-pot” reaction. By the installing of a cyano-group on pyridine scaffold, the proton donating ability of -NH2 was greatly enhanced, enabling 2-APC to undergo ESIPT-e process. Surprisingly, 2-APC exhibited dual-emissions in protic solvents ethanol and normal fluorescence in aprotic solvents, which is vastly different from that of conventional ESIPT-a dyes. The ESIPT emission can be obviously suppressed by Fe3+ due to the coordination reaction of Fe3+ with the A-D system in 2-APC. From this basis, a highly sensitive and selective method was established using 2-APC as a fluorescent probe, which offers the sensitive detection of Fe3+ ranging from 0 to 13 μM with the detection limit of 7.5 nM. The recovery study of spiked Fe3+ measured by the probe showed satisfactory results (97.2103.4%) with the reasonable RSD ranging from 3.1 to 3.8%. Moreover, 2-APC can also exhibit aggregation-induced effect in poor solvent or solid-state, eliciting strong red fluorescence. 2-APC was also applied to cell-imaging, exhibiting good cell-permeability, biocompatibility and color rendering. This multi-mode emission of 2-APC is significant departure from that of conventional extended p-conjugated systems and ESIPT dyes based on a flat and rigid molecular design. The “one-pot synthesis” strategy for the construction of ESIPT molecules pioneered a new route to achieve tricolor-emissive fluorophores.

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.

In-depth NMR and IR study of the proton transfer equilibrium between [{(MeC(CH2PPh2)3}Ru(CO)H2] and hexafluoroisopropanol

2001 ◽  
Vol 79 (5-6) ◽  
pp. 479-489 ◽  
Author(s):  
Vladimir I Bakhmutov ◽  
Ekaterina V Bakhmutova ◽  
Natalia V Belkova ◽  
Claudio Bianchini ◽  
Lina M Epstein ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Ir Spectroscopy ◽  
Proton Transfer ◽  
Proton Transfer Reaction ◽  
Proton Donor ◽  
Strong Hydrogen Bond ◽  
Nmr Study ◽  
Ir Study

The (carbonyl)dihydride complex [(triphos)Ru(CO)H2] (2) has been synthesized by reaction of the ruthenate [(triphos)RuH3]K (triphos = MeC(CH2PPh2)3) with ethanol saturated with CO. A single crystal X-ray analysis and IR and NMR experiments have shown that 2 adopts in both the solid state and solution an octahedral coordination geometry with a facial triphos ligand, two cis terminal hydrides, and a terminal carbonyl. The reaction of hexafluoro-2-propanol (HFIP) with 2 has been studied in CH2Cl2 solution by IR and NMR spectroscopy. The proton donor interacts with a terminal hydride of 2 forming a rather strong hydrogen bond. The resulting H-bonded adduct [{(triphos)Ru(CO)(H)H}···{HOCH(CF3)2}] (2a) has fully been characterized by in situ NMR and IR techniques. Compound 2a is in equilibrium with the nonclassical η2-H2 complex [(triphos)Ru(CO)H(H2)]+ (2b), which can independently be prepared by protonation of 2 with a strong protic acid at low temperature. Unequivocal characterization of the dihydrogen complex (2b) has been achieved by a multifaceted spectroscopic investigation (Tobs1min = 0.005 s (200 MHz), JH,D [Formula: see text] 30 Hz, DQCC = 78.3 kHz). A combined IR and NMR study of the proton transfer reaction involving 2 and HFIP in CH2Cl2 to give, first, the H-bonded adduct (2a) and, then, the dihydrogen complex (2b) has demonstrated that all these species are in equilibrium in the temperature range from 190 to 260 K. The thermodynamic parameters for the formation of 2a have independently been determined by NMR and IR methods, while those for the formation of 2b have been obtained by IR spectroscopy. An energetic profile for the reaction sequence 2 [Formula: see text] 2a [Formula: see text] 2b is proposed and discussed.Key words: hydrides, hydrogen bonding, ruthenium, IR spectroscopy, NMR spectroscopy.

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