Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme |
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Authors: | Trujillo M; Duncan R; Santi DV |
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Affiliation: | Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA. |
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Abstract: | A gene encoding a bifunctional homodimeric dihydrofolate reductase-
thymidylate synthase (DHFR-TS) was constructed by destroying the stop codon
of Escherichia coli dihydrofolate reductase (DHFR) and joining the coding
sequences of the monofunctional enzymes by a five amino acid linker. The
protein was designed to mimic features of active site proximity and
electrostatics in the protozoan DHFR-TSs which are believed to be important
in channeling of the DHFR substrate, H2folate, to TS. The genetically
engineered catalytically active homodimeric bifunctional DHFR-TS was
expressed, purified and characterized. The component activities of the
purified bifunctional enzyme had kinetic properties similar to those of the
monofunctional TS and DHFR, but unlike the authentic bifunctional enzymes
from protozoa this enzyme did not kinetically channel dihydrofolate from
DHFR to TS.
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