scholarly journals Oscillatory Active-Site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase

ACS Catalysis ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 11199-11206 ◽  
Author(s):  
Philip Pagano ◽  
Qi Guo ◽  
Chethya Ranasinghe ◽  
Evan Schroeder ◽  
Kevin Robben ◽  
...  
Keyword(s):  
Active Site ◽  
Formate Dehydrogenase ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope

QM/MM calculations of kinetic isotope effects in the chorismate mutase active site

2003 ◽  
Vol 1 (3) ◽  
pp. 483-487 ◽  
Author(s):  
Sergio Martí ◽  
Vicent Moliner ◽  
Iñaki Tuñón ◽  
Ian H. Williams
Keyword(s):  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Chorismate Mutase ◽  
Kinetic Isotope

scholarly journals Kinetic Isotope Effects for Alkaline Phosphatase Reactions:  Implications for the Role of Active-Site Metal Ions in Catalysis

2007 ◽  
Vol 129 (31) ◽  
pp. 9789-9798 ◽  
Author(s):  
Jesse G. Zalatan ◽  
Irina Catrina ◽  
Rebecca Mitchell ◽  
Piotr K. Grzyska ◽  
Patrick J. O'Brien ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Kinetic Isotope

scholarly journals Temperature dependence of dynamic, tunnelling and kinetic isotope effects in formate dehydrogenase

2018 ◽  
Vol 20 (40) ◽  
pp. 25722-25737 ◽  
Author(s):  
Maite Roca ◽  
J. Javier Ruiz-Pernía ◽  
Raquel Castillo ◽  
Mónica Oliva ◽  
Vicent Moliner
Keyword(s):  
Temperature Dependence ◽  
Chemical Reaction ◽  
Formate Dehydrogenase ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Chemical System ◽  
Kinetic Isotope

The protein cannot be considered as a passive spectator of the chemical system; it is part of the chemical reaction.

scholarly journals Kinetic isotope effects and ligand binding in PQQ-dependent methanol dehydrogenase

2005 ◽  
Vol 388 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Parvinder HOTHI ◽  
Michael J. SUTCLIFFE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Steady State ◽  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Competitive Binding ◽  
Catalytic Site ◽  
Methanol Dehydrogenase ◽  
Ammonium Concentration ◽  
Steric Interaction ◽  
Kinetic Isotope

The reaction of PQQ (2,7,9-tricarboxypyrroloquinoline quinone)-dependent MDH (methanol dehydrogenase) from Methylophilus methylotrophus has been studied under steady-state conditions in the presence of an alternative activator [GEE (glycine ethyl ester)] and compared with similar reactions performed with ammonium (used more generally as an activator in steady-state analysis of MDH). Studies of initial velocity with methanol (protiated methanol, C1H3O1H) and [2H]methanol (deuteriated methanol, C2H3O2H) as substrate, performed with different concentrations of GEE and PES (phenazine ethosulphate), indicate competitive binding effects for substrate and PES on the stimulation and inhibition of enzyme activity by GEE. GEE is more effective at stimulating activity than ammonium at low concentrations, suggesting tighter binding of GEE to the active site. Inhibition of activity at high GEE concentration is less pronounced than at high ammonium concentration. This suggests a close spatial relationship between the stimulatory (KS) and inhibitory (KI) binding sites in that binding of GEE to the KS site sterically impairs the binding of GEE to the KI site. The binding of GEE is also competitive with the binding of PES, and GEE is more effective than ammonium in competing with PES. This competitive binding of GEE and PES lowers the effective concentration of PES at the site competent for electron transfer. Accordingly, the oxidative half-reaction, which is second-order with respect to PES concentration, is more rate-limiting in steady-state turnover with GEE than with ammonium. The smaller methanol C-1H/C-2H kinetic isotope effects observed with GEE are consistent with a larger contribution made by the oxidative half-reaction to rate limitation. Cyanide is much less effective at suppressing ‘endogenous’ activity in the presence of GEE than with ammonium, which is attributed to impaired binding of cyanide to the catalytic site through steric interaction with GEE bound at the KS site. The kinetic model developed previously for reactions of MDH with ammonium [Hothi, Basran, Sutcliffe and Scrutton (2003) Biochemistry 42, 3966–3978] is consistent with data obtained with GEE, although a more detailed structural interpretation is given here. Molecular-modelling studies rationalize the kinetic observations in terms of a complex binding scenario at the molecular level involving two spatially distinct inhibitory sites (KI and KI′). The KI′ site caps the entrance to the active site and is interpreted as the PES binding site. The KI site is adjacent to, and, for GEE, overlaps with, the KS site, and is located in the active-site cavity close to the PQQ cofactor and the catalytic site for methanol oxidation.

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.

Kinetic isotope effects in the active site ofB. subtilis chorismate mutase

2003 ◽  
Vol 94 (5) ◽  
pp. 287-292 ◽  
Author(s):  
Sharon E. Worthington ◽  
Adrian E. Roitberg ◽  
Morris Krauss
Keyword(s):  
Active Site ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Chorismate Mutase ◽  
Kinetic Isotope
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