Publication

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

Swanwick, RS., Maglia, G., Tey, LH. & Allemann, RK., Feb-2006, In : Biochemical Journal. 394, p. 259-265 6 p.

Research output: Contribution to journalArticleAcademicpeer-review

APA

Swanwick, RS., Maglia, G., Tey, LH., & Allemann, RK. (2006). Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase. Biochemical Journal, 394, 259-265.

Author

Swanwick, RS ; Maglia, Giovanni ; Tey, LH ; Allemann, RK. / Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase. In: Biochemical Journal. 2006 ; Vol. 394. pp. 259-265.

Harvard

Swanwick, RS, Maglia, G, Tey, LH & Allemann, RK 2006, 'Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase', Biochemical Journal, vol. 394, pp. 259-265.

Standard

Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase. / Swanwick, RS; Maglia, Giovanni; Tey, LH; Allemann, RK.

In: Biochemical Journal, Vol. 394, 02.2006, p. 259-265.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

Swanwick RS, Maglia G, Tey LH, Allemann RK. Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase. Biochemical Journal. 2006 Feb;394:259-265.


BibTeX

@article{d65ca104dcee49e1ad9120dc0986d8f8,
title = "Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase",
abstract = "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 oil 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 degrees C to 2.2 +/- 0.2 at 40 degrees 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, Gly(121), with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121 V-DHFR, in which Gly(121) was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G 121 V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121 V-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 degrees 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.",
keywords = "Hydride transfer, enzyme catalysis, active-site, soybean lipoxygenase-1, promoting vibration, transfer rates, dynamics, mechanism, loop, flexibility",
author = "RS Swanwick and Giovanni Maglia and LH Tey and RK Allemann",
year = "2006",
month = "2",
language = "English",
volume = "394",
pages = "259--265",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "PORTLAND PRESS LTD",

}

RIS

TY - JOUR

T1 - Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase

AU - Swanwick, RS

AU - Maglia, Giovanni

AU - Tey, LH

AU - Allemann, RK

PY - 2006/2

Y1 - 2006/2

N2 - 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 oil 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 degrees C to 2.2 +/- 0.2 at 40 degrees 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, Gly(121), with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121 V-DHFR, in which Gly(121) was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G 121 V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121 V-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 degrees 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.

AB - 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 oil 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 degrees C to 2.2 +/- 0.2 at 40 degrees 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, Gly(121), with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121 V-DHFR, in which Gly(121) was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G 121 V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121 V-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 degrees 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.

KW - Hydride transfer

KW - enzyme catalysis

KW - active-site

KW - soybean lipoxygenase-1

KW - promoting vibration

KW - transfer rates

KW - dynamics

KW - mechanism

KW - loop

KW - flexibility

M3 - Article

VL - 394

SP - 259

EP - 265

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -

ID: 36008408