Thymidine kinase is a key enzyme in the nucleotide salvage pathway,
catalyzing the production of dTMP from thymidine and ATP. In order to
identify the structural features of the TK protein and/or primary amino
acid sequences which contribute to the catalytic and regulatory activities of
this enzyme, an in vitro transcription and translation system has been used
in concert with protein engineering techniques for the production and
phenotypic characterization of mutant and wild-type TK enzymes. Because
of discrepancies in the literature regarding the quaternary structure of the
VVTK, the native molecular weight and quaternary structure was
determined to be an 80kDa homotetrameric enzyme by glycerol gradient
fractionation, gel filtration and glutaraldehyde cross-linking analyses.
Computer-assisted alignment of the predicted amino acid sequences
derived from cellular and poxvirus TK genes identified seven highly-conserved
domains distributed throughout the VVTK polypeptide
(domains I-VII). Domain I (amino acid residues 11-18 ) exhibits a high
degree of similarity to both ATP and GTP binding site consensus sequences,
although the VVTK utilizes only ATP as a phosphate donor. Site directed
mutagenesis and ATP-agarose affinity chromatography techniques were
employed to confirm that this region was responsible for ATP binding and
to determine which amino acids were essential for efficient binding.
The TK gene (tdk) from E. coli was isolated and sequenced to serve as
a prokaryotic enzyme with which to compare VVTK. The alignment
revealed only 23% shared identity with VVTK and, interestingly, the
identical and similar residues were clustered into three of the seven
domains identified previously (domains I, III and VII).
Preliminary evidence supports domain III (residues 78-90) as a
putative magnesium binding site and that a highly conserved cysteine
residue (cysteine 170) within domain VII (residues 168-171) may be a
component of the catalytic site. Secondary structure alignment between
Herpes Simplex Virus (HSV) TK and monkeypox TK (a close relative of
VVTK) revealed that the putative nucleoside binding site of HSVTK aligns
with residues within domain IV. Replacement of a VVTK residue (Q114)
with the corresponding residue of HSVTK (an aspartic acid) greatly alters
the substrate specificity and dTTP sensitivity of VVTK. / Graduation date: 1991
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/37105 |
| Date | 30 April 1991 |
| Creators | Black, Margaret E. |
| Contributors | Hruby, Dennis E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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