This thesis presents an investigation of the hydrogen transfer reactions between dihydrofolate (H2F) and NADPH that are catalysed by the dihydrofolate reductase (DHFR) isolated from Geobacillus stearothermophilus (BsDHFR) as well as an artificial hybrid originating from the DHFRs from mesophilic Escherichia coli (EcDHFR) and hyperthermophilic Thermotoga maritima (TmDHFR). A broad spectrum of studies, focusing on the relationship between structure, thermostability and kinetics, showed that the catalytic behaviour of BsDHFR is generally similar to other monomeric DHFRs, including ones found in the mesophile Escherichia coli and the psychrophile Moritella profunda, but significantly different from the dimeric TmDHFR. The fact that all monomeric DHFRs display similar catalytic behaviour, regardless of their widely different optimal temperatures, suggests that thermostability does not directly relate to catalytic efficiency. The biophysical differences between monomeric DHFRs and TmDHFR are likely derived from the dimeric nature of the hyperthermophilic enzyme. An artificial dimeric variant of EcDHFR, Xet-3, was prepared by introducing residues at the dimer interface of TmDHFR. While thermostability of this variant is enhanced, it showed a great decrease in its steady-state and pre-steady-state rate constants. Given that the corresponding rate constants did not increase when the loops are released in the monomeric variant of TmDHFR, the lowered catalytic ability in Xet-3 is likely a consequence of geometric distortion of the active site and loss of loop flexibility that is catalytically important in EcDHFR. In contrast, the relatively poor activity of TmDHFR is not simply a consequence of reduced loop flexibility; the dimer interface of TmDHFR plays a rather complicated role in catalysis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:585347 |
| Date | January 2013 |
| Creators | Guo, Jian Nan |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/56290/ |
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