pH Dependence of Enzyme Reaction Rates and Deuterium Isotope Effects on the Reduction of a New Mechanism-Based Substrate by Dihydrofolate Reductase (DHFR)

Biochemistry ◽  
1995 ◽  
Vol 34 (11) ◽  
pp. 3734-3741 ◽  
Author(s):  
Soon-Seog Jeong ◽  
Jill E. Gready
Keyword(s):  
Dihydrofolate Reductase ◽  
Isotope Effects ◽  
Ph Dependence ◽  
Enzyme Reaction ◽  
Reaction Rates ◽  
Deuterium Isotope Effects

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.

Hydrocarbon C-H bond activation by rhodium porphyrins

2004 ◽  
Vol 08 (02) ◽  
pp. 103-110 ◽  
Author(s):  
Weihong Cui ◽  
Bradford B. Wayland
Keyword(s):  
Transition State ◽  
High Selectivity ◽  
Bond Activation ◽  
Isotope Effects ◽  
Reaction Rates ◽  
Methane Activation ◽  
Porphyrin Ligand ◽  
Deuterium Isotope Effects ◽  
Rhodium Porphyrins ◽  
Steric Constraints

Rhodium porphyrins provide a variety of C-H bond reactions with both aromatic and aliphatic hydrocarbons that acquire unusual selectivity in part through the steric requirements of the porphyrin ligand. Rhodium(III) porphyrins selectively react with aromatic C-H bonds by electrophilic substitution with the virtual exclusion of aliphatic C-H bond activation. Rhodium(II) porphyrins react by a metal-centered radical pathway with alkyl aromatics and alkanes selectively at the alkyl C-H bond with total exclusion of aromatic C-H bond activation. Reactions of rhodium(II) metalloradicals with alkyl C-H bonds have large deuterium isotope effects, small activation enthalpies and large negative activation entropies consistent with a near linear symmetrical four-centered transition state ( Rh ˙⋯ H ⋯ C ⋯˙Rh). The nature of this transition state and the dimensions of rhodium porphyrins provide steric constraints that preclude aromatic C-H bond reactions and give high kinetic preference for methane activation as the smallest alkane substrate. Rhodium(II) tethered diporphyrin bimetalloradical complexes convert the C-H bond reactions to bimolecular processes with dramatically increased reaction rates and high selectivity for methane activation.

scholarly journals Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase

2006 ◽  
Vol 394 (1) ◽  
pp. 259-265 ◽  
Author(s):  
Richard S. Swanwick ◽  
Giovanni Maglia ◽  
Lai-hock Tey ◽  
Rudolf K. Allemann
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Hydrogen Transfer ◽  
Isotope Effects ◽  
Hydride Transfer ◽  
Kinetic Isotope Effects ◽  
Reaction Rates ◽  
Protein Motions ◽  
Kinetic Isotope ◽  
Exponential Factors

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0±0.2 at 5 °C to 2.2±0.2 at 40 °C and an inverse ratio of the pre-exponential factors of 0.108±0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue, Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 °C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.

Reactions of alicyclic ketones in carbon tetrachloride. I, The kinetics of the chlorination of cyclopentanone and cyclohexanone catalyzed by hydrogen chloride

1986 ◽  
Vol 64 (9) ◽  
pp. 1681-1689 ◽  
Author(s):  
Eize J. Stamhuis ◽  
Henk Maatman ◽  
Henk Stinissen ◽  
Geert E. H. Joosten
Keyword(s):  
Carbon Tetrachloride ◽  
Hydrogen Chloride ◽  
Isotope Effects ◽  
Reaction Rates ◽  
Zero Order ◽  
Rate Determining Step ◽  
Conjugate Acid ◽  
Deuterium Isotope Effects ◽  
Self Association ◽  
Kinetics Of

The kinetics of the direct chlorination of cyclopentanone (cp) and cyclohexanone (ch) in carbon tetrachloride, catalyzed by hydrogen chloride, was studied. The rate of chlorination, measured by flow and stopped-flow techniques, is zero order in chlorine; the order in cp and ch increases from 1 at [cp] and [ch] of 0.01 M concentration to 2 at concentrations of 1 M. This is explained by self-association of the ketones in carbon tetrachloride solutions. The order in hydrogen chloride is 1. Since this compound is one of the products, the reaction is autocatalytic. Deuterium isotope effects and the kinetic data strongly point to a mechanism in which the oxygen-protonated monomeric ketone is α-carbon deprotonated in a rate-determining step. This step, which is catalyzed by the bases cp or ch, respectively, leads to the corresponding enol as intermediate. The enol is then chlorinated very rapidly. In addition to the chloro ketone, very reactive chloride anions are formed. A small fraction of these anions deprotonate α- or α′-carbon atoms of the oxygen-conjugate acid of the monochloro ketone. The remainder are captured by HCl to form energetically more favored Cl--(HCl)n complexes with n = 1, 2, or 3. This explains why, even at low conversions of the ketones, substantial amounts of the various dichloro isomers are formed in addition to monochloro products. A rate expression is derived, which excellently describes the experimentally obtained rates of chlorination of cp and ch over a range of reaction rates of more than three decades.

Mechanism of hydration and isomerisation of 4-ethylidene-2,6-di-tert-butyl-2,5-cyclohexadien-1-one. Kinetics, acid-base catalysis and solvent deuterium isotope effects

1979 ◽  
Vol 44 (1) ◽  
pp. 110-122 ◽  
Author(s):  
Jiří Velek ◽  
Bohumír Koutek ◽  
Milan Souček
Keyword(s):  
Aqueous Medium ◽  
Rate Equation ◽  
Kinetic Data ◽  
Isotope Effects ◽  
Base Catalysis ◽  
Quinone Methide ◽  
Reaction Products ◽  
Acid Base ◽  
Deuterium Isotope Effects ◽  
Hydroxybenzyl Alcohol

Competitive hydration and isomerisation of the quinone methide I at 25 °C in an aqueous medium in the region of pH 2.4-13.0 was studied spectrophotometrically. The only reaction products in the studied range of pH are 4-hydroxybenzyl alcohol (II) and 4-hydroxystyrene (III). The form of the overall rate equation corresponds to a general acid-base catalysis. The mechanism of both reactions for three markedly separated pH regions is discussed on the basis of kinetic data and solvent deuterium effect.

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