Coupling Interactions of Distal Residues Enhance Dihydrofolate Reductase Catalysis:  Mutational Effects on Hydride Transfer Rates†

Biochemistry ◽  
2002 ◽  
Vol 41 (42) ◽  
pp. 12618-12628 ◽  
Author(s):  
P. T. Ravi Rajagopalan ◽  
Stefan Lutz ◽  
Stephen J. Benkovic
Keyword(s):  
Dihydrofolate Reductase ◽  
Hydride Transfer ◽  
Transfer Rates

scholarly journals Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima

2003 ◽  
Vol 374 (2) ◽  
pp. 529-535 ◽  
Author(s):  
Giovanni MAGLIA ◽  
Masood H. JAVED ◽  
Rudolf K. ALLEMANN
Keyword(s):  
Steady State ◽  
Dihydrofolate Reductase ◽  
Tertiary Structure ◽  
Thermotoga Maritima ◽  
Hydride Transfer ◽  
Kinetic Scheme ◽  
Transfer Rates ◽  
E Coli ◽  
Steady State Kinetics ◽  
Rate Limiting

DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally. Its tertiary structure is similar to that of DHFR from E. coli but it is the only DHFR characterized so far that relies on dimerization for stability. The midpoint of the thermal unfolding of TmDHFR was at approx. 83 °C, which was 30 °C higher than the melting temperature of DHFR from E. coli. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy. The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution. Hydride transfer was slow (0.14 s−1 at 25 °C) and at least partially rate limiting at low temperatures but increased dramatically with temperature. At 80 °C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E. coli enzyme at its physiological temperature. Hydride transfer depended on ionization of a single group in the active site with a pKa of 6.0. While at 30 °C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E. coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures.

scholarly journals The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

2006 ◽  
Vol 361 (1472) ◽  
pp. 1307-1315 ◽  
Author(s):  
Lin Wang ◽  
Nina M Goodey ◽  
Stephen J Benkovic ◽  
Amnon Kohen
Keyword(s):  
Temperature Dependence ◽  
Dihydrofolate Reductase ◽  
Isotope Effects ◽  
Hydride Transfer ◽  
Activation Parameters ◽  
Physical Nature ◽  
Enzyme Dynamics ◽  
Temperature Dependences ◽  
Donor Acceptor ◽  
Significant Temperature

Residues M42 and G121 of Escherichia coli dihydrofolate reductase ( ec DHFR) are on opposite sides of the catalytic centre (15 and 19 Å away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ec DHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor–acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface pre-arrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis

Biochemistry ◽  
1991 ◽  
Vol 30 (49) ◽  
pp. 11567-11579 ◽  
Author(s):  
Martin F. Farnum ◽  
Douglas Magde ◽  
Elizabeth E. Howell ◽  
John T. Hirai ◽  
Mark S. Warren ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Hydride Transfer ◽  
Fluorescence Decay ◽  
Molecular Motions

scholarly journals Extension and Limits of the Network of Coupled Motions Correlated to Hydride Transfer in Dihydrofolate Reductase

2014 ◽  
Vol 136 (6) ◽  
pp. 2575-2582 ◽  
Author(s):  
Priyanka Singh ◽  
Arundhuti Sen ◽  
Kevin Francis ◽  
Amnon Kohen
Keyword(s):  
Dihydrofolate Reductase ◽  
Hydride Transfer
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