0000000000672934
AUTHOR
E. Joel Loveridge
Increased dynamic effects in a catalytically compromised variant of Escherichia coli dihydrofolate reductase
Isotopic substitution (15N, 13C, 2H) of a catalytically compromised variant of Escherichia coli dihydrofolate reductase, EcDHFR-N23PP/S148A, has been used to investigate the effect of these mutations on catalysis. The reduction of the rate constant of the chemical step in the EcDHFR-N23PP/S148A catalyzed reaction is essentially a consequence of an increase of the quasi-classical free energy barrier and to a minor extent of an increased number of recrossing trajectories on the transition state dividing surface. Since the variant enzyme is less well set up to catalyze the reaction, a higher degree of active site reorganization is needed to reach the TS. Although millisecond active site motion…
Unraveling the role of protein dynamics in dihydrofolate reductase catalysis
Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy ((15)N, (13)C, (2)H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy e…
Protein isotope effects in dihydrofolate reductase from Geobacillus stearothermophilus show entropic-enthalpic compensatory effects on the rate constant.
Catalysis by dihydrofolate reductase from the moderately thermophilic bacterium Geobacillus stearothermophilus (BsDHFR) was investigated by isotope substitution of the enzyme. The enzyme kinetic isotope effect for hydride transfer was close to unity at physiological temperatures but increased with decreasing temperatures to a value of 1.65 at 5 °C. This behavior is opposite to that observed for DHFR from Escherichia coli (EcDHFR), where the enzyme kinetic isotope effect increased slightly with increasing temperature. These experimental results were reproduced in the framework of variational transition-state theory that includes a dynamical recrossing coefficient that varies with the mass of…