Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Five thymidine kinase (TK)-deficient mutants (B1 to B5) of bovine herpesvirus type 1 (BHV-1) were isolated by selection for resistance to the nucleotide analogue bromovinyldeoxyuridine. The genetic lesion in mutant B1 was localized in a 2.7 kb SalI-SalI subfragment (fTK2.7) which maps between 0.456 and 0.475 within the HindIII A fragment of the BHV-1 genome. The tk genes from wild-type and the TK-mutants B1 to B5 were cloned and sequenced using eight unique synthetic primers designed from a published sequence. The BHV-1 tk gene sequence for the strain 6660 contained some differences compared with that published previously for strain LA. Alignment of the predicted amino acid sequence of the BHV-1 TK polypeptide with different herpesvirus TKs revealed five strongly conserved regions and also identified putative functional relationships with other enzymes. Several interesting features were apparent in the tk gene sequences from the TK- mutants. The TK mutant B1 was a typical frameshift and chain termination mutant due to the deletion of a single base. The tk gene sequence of mutant B2 revealed the deletion of three bases resulting in the loss of valine at amino acid residue 174 of the TK polypeptide. The tk genes of mutants B3 to B5 contained an identical change of a single base addition resulting in frameshift and premature chain termination. In contrast to wild-type BHV-1, the TK-defective mutants were incapable of adsorbing TK-neutralizing antibodies from serum.
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PMID:Analysis of the bovine herpesvirus type 1 thymidine kinase (TK) gene from wild-type virus and TK-deficient mutants. 254 66

The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Km's of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Km's of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.
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PMID:Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. 283 27

Several VV structural proteins are produced by the removal of amino-terminal peptides from their cognate precursors. In the experiments reported here, directed genetic approaches were used to investigate the possible role of these terminal peptides in protein processing. As a model system, the FLAG epitope-tagged P25K precursor was used to prepare constructs in which the 31-amino-acid P25K N-terminal peptide was removed or replaced by heterologous sequences, while the -A-G*-A- cleavage motif was retained. Only a trace amount of the leaderless P25KF(delta 31) polypeptide was found within the mature virions, implying that proteolytic processing is necessary for the incorporation of the 25K product into mature virions. In trans-processing assays, significant levels of the 25K product were generated from wild-type P25KF and P4b:25KF, which consists of the 61-amino-acid P4b terminal peptide, and from P4b:25KF with 15, 30, or 44 residues of the amino terminus deleted. In contrast, only a small amount of 25K was produced from the TK:25KF, which contains the amino-terminal 30 residues of VV thymidine kinase, a protein which is not cleaved under normal circumstances. Furthermore, it has been hypothesized that a hydrophobic residue is required at position P4 relative to the -A-G*-A- motif for the cleavage to take place. An intermediate level of the 25K product was detected from the TK:25KF(Q29V) mutant which has the glutamine residue at P4 replaced with a valine residue, suggesting that the hydrophobic P4 residue and additional substrate determinants in the N-terminal peptide region are required for the proteolytic processing reaction to occur normally. Taken together, these data suggest that the amino-terminal peptides of the VV core proteins are to some extent interchangeable and that the residues proximal to the AGA site are of most importance.
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PMID:Analysis of the role of the amino-terminal peptide of vaccinia virus structural protein precursors during proteolytic processing. 787 30

Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.
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PMID:Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties. 1092 19

There is no effective therapy for hormone-refractory prostate cancer and a novel therapeutic modality, such as a gene therapy, should be actively pursued. Previously, Gardner and Chung conducted a phase I clinical trial of Ad-OC-TK (recombinant adenoviral vector containing osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) for the treatment of hormone-refractory prostate cancer at the University of Virginia. We report on our ongoing phase I/II clinical trial of Ad-OC-TK plus VAL for the treatment of advanced prostate cancer at the Kobe University Hospital, Japan.
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PMID:Progress report on phase I/II clinical trial of Ad-OC-TK plus VAL therapy for metastatic or locally recurrent prostate cancer: Initial experience at Kobe University. 1683 76

We evaluated the long-term safety and efficacy of Ad-OC-TK (recombinant adenoviral vector carrying an osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) gene therapy for hormone-refractory prostate cancer. Ad-OC-TK/VAL therapy is the first in vivo adenovirus-mediated gene therapy to be used to treat metastatic prostate cancer, including bone metastasis. Six patients were enrolled in this trial, and two doses of Ad-OC-TK (2.5 x 10(9) or 2.5 x 10(10) plaque-forming units) were injected into locally recurrent tumor or bone metastasis on day 1 and day 8. Patients were also given VAL (3 g/day) for 21 days. Safety and efficacy were evaluated for at least 8 months in each patient. All patients tolerated this therapy with no serious adverse events. One prostate-specific antigen (PSA) response (from 318.3 to 4.9 ng/ml) was observed with a time to PSA progression (TTP) of 12 months. Docetaxel (30 mg/m2 per week) and estramustine (560 mg/day) combination chemotherapy (DE) was given to three docetaxel-naive patients on PSA failure after gene therapy. All three patients had a PSA response to DE therapy with 21, 7, and 4 months of TTP. These results suggest that additional trials are warranted.
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PMID:Long-term outcome of phase I/II clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer. 1802 Oct 19