6533b871fe1ef96bd12d265a

RESEARCH PRODUCT

Protein isotope effects in dihydrofolate reductase from Geobacillus stearothermophilus show entropic-enthalpic compensatory effects on the rate constant.

E. Joel LoveridgeIñaki TuñónVicent MolinerLouis Y. P. LukJ. Javier Ruiz-perníaRudolf Konrad AllemannWilliam M. Dawson

subject

Models MolecularRate constantsStatic ElectricityDihydrofolate reductaseMolecular ConformationThermodynamicsBiochemistryCatalysisCatalysisModerately thermophilicGeobacillus stearothermophilusColloid and Surface ChemistryReaction rate constantDihydrofolate reductaseKinetic isotope effectEscherichia coliGeobacillus stearothermophilusQDTransmission coefficientIncreasing temperaturesCarbon IsotopesbiologyIsotopeNitrogen IsotopesHydrideChemistryKinetic isotope effectsGeneral ChemistryCrystallographyTetrahydrofolate Dehydrogenasebiology.proteinThermodynamics

description

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 the protein. Our simulations indicate that BsDHFR has greater flexibility than EcDHFR on the ps–ns time scale, which affects the coupling of the environmental motions of the protein to the chemical coordinate and consequently to the recrossing trajectories on the reaction barrier. The intensity of the dynamic coupling in DHFRs is influenced by compensatory temperature-dependent factors, namely the enthalpic barrier needed to achieve an ideal transition-state configuration with minimal nonproductive trajectories and the protein disorder that disrupts the electrostatic preorganization required to stabilize the transition state. Together with our previous studies of other DHFRs, the results presented here provide a general explanation why protein dynamic effects vary between enzymes. Our theoretical treatment demonstrates that these effects can be satisfactorily reproduced by including a transmission coefficient in the rate constant calculation, whose dependence on temperature is affected by the protein flexibility. The authors acknowledge computational resources from the University of Valencia (Tirant supercomputer) and from University Jaume I. This work was supported by grants BB/L020394/1 and BB/J005266/1 (RKA) from the UK Biotechnology and Biological Sciences Research Council (BBSRC) and EP/L027240/1 from the UK Engineering and Physical Sciences Research Council (EPSRC), by FEDER and Ministerio de Economía y Competitividad funds (project CTQ2012-36253-C03), Generalitat Valenciana (ACOMP/2014/277 and PrometeoII/2014/022) and by Universitat Jaume I (Project P1·1B2011–23).

10.1021/ja5102536https://pubmed.ncbi.nlm.nih.gov/25396728