The role for protein dynamics in the transition states (TS) of enzyme reactions has been debated over decades. Dihydrofolate reductase (DHFR) catalyses the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate and has long been considered a paradigm of enzymology. Numerous studies on DHFR have provided strong evidence that there is no coupling between the long-range motions on nanosecond to millisecond timescales to the chemical coordinate. However, the role of femtosecond (fs) bond vibrations in the TS has not been fully investigated. This investigation focused on understanding how these fast protein vibrations affect enzyme catalysis. A thermophilic DHFR from Geobacillus stearothermophilus (BsDHFR) was investigated kinetically by complete enzyme isotope substitution. Our studies indicated that, whilst protein vibrations do couple to the reaction coordinate, they do not affect the height or width of barrier crossing. Instead, dynamic coupling enhances the frequency of dynamic recrossing. In line with the other DHFR investigations by enzyme isotope substitution, efficient enzymes tend to reduce dynamic coupling as a mean to maximise enzyme catalytic efficiency. The transition state in DHFR was also characterised by α-secondary hydrogen and heavy atom kinetic isotope effects (KIEs). Secondary KIEs were measured for DHFR isolated from Escherichia coli, Moritella profunda and G. stearothermophilus. The high resemblance in the magnitude and temperature dependence of the measured α-secondary KIE implied that the reaction ready configuration is essentially the same among these enzymes. Carbon isotope effect measurements were measured for EcDHFR. The reacting carbon in NADPH showed a profound isotope effect at low temperature. Further analysis by measuring hydride KIE indicated the hypothetical "promoting" motion is unlikely to act via the C4 atom of NADPH. The carbon isotope effect likely reports on the recrossing events or the reorganisation effect that occur along the transition state dividing surface.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:633562 |
| Date | January 2014 |
| Creators | Dawson, William |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/68719/ |
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