Thermophilicity and catalytic efficiency in dihydrofolate reductase
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Abstract
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