Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [3H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.
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PMID:Active-site modification of mammalian DNA polymerase beta with pyridoxal 5'-phosphate: mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket. 250 25

DNA polymerase alpha from Drosophila melanogaster embryos is a multisubunit enzyme complex which can exhibit DNA polymerase, 3'----5' exonuclease, and DNA primase activities. Pyridoxal 5'-phosphate (PLP) inhibition of DNA polymerase activity in this complex is time dependent and exhibits saturation kinetics. Inhibition can be reversed by incubation with an excess of a primary amine unless the PLP-enzyme conjugate is first reduced with NaBH4. These results indicate that PLP inhibition occurs via imine formation at a specific site(s) on the enzyme. Results from substrate protection experiments are most consistent with inhibition of DNA polymerase activity by PLP binding to either one of two sites. One site (PLP site 1) can be protected from PLP inhibition by any nucleoside triphosphate in the absence or presence of template-primer, suggesting that PLP site 1 defines a nucleotide-binding site which is important for DNA polymerase activity but which is distinct from the DNA polymerase active site. PLP also inhibits DNA primase activity of the DNA polymerase alpha complex, and primase activity can be protected from PLP inhibition by nucleotide alone, arguing that PLP site 1 lies within the DNA primase active site. The second inhibitory PLP-binding site (PLP site 2) is only protected from PLP inhibition when the enzyme is bound to both template-primer and correct dNTP in a stable ternary complex. Since binding of PLP at site 2 is mutually exclusive with template-directed dNTP binding at the DNA polymerase active site, PLP site 2 appears to define the dNTP binding domain of the active site. Results from initial velocity analysis of PLP inhibition argue that there is a rate-limiting step in the polymerization cycle during product release and/or translocation.
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PMID:Affinity labeling the DNA polymerase alpha complex. I. Pyridoxal 5'-phosphate inhibition of DNA polymerase and DNA primase activities of the DNA polymerase alpha complex from Drosophila melanogaster embryos. 313 61

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.
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PMID:Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site. 314 25

Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with sodium [3H]borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.
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PMID:Site-specific modification of Escherichia coli DNA polymerase I large fragment with pyridoxal 5'-phosphate. 642 12

Pyridoxal phosphate modification of adenovirus DNA polymerase results in loss of DNA polymerase activity, whereas the 3' --> 5' exonuclease activity is unaffected. Inhibition by pyridoxal phosphate is time-dependent, displays saturation kinetics, and is reversible in the presence of excess primary amine unless the pyridoxal phosphate-enzyme adduct is first reduced with NaBH4. Thus, inhibition is the consequence of Schiff base formation between the aldehyde moiety of pyridoxal phosphate and primary amino groups on the enzyme. In addition to inhibiting DNA polymerase activity, pyridoxal phosphate also inhibited the ability of the enzyme to initiate viral DNA replication, by transfer of dCMP onto the preterminal protein. Neither template-primer nor dNTP protect against pyridoxal phosphate inhibition, but the combination of template-primer and complementary substrate dNTP protected both initiation and DNA polymerase activities. Thus, it is likely that both the dCMP transfer activity required for initiation and DNA polymerase activity are carried out at the same site of the enzyme.
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PMID:Pyridoxal 5'-phosphate inhibition of adenovirus DNA polymerase. 879 69

Vitamin B(6) compounds such as pyridoxal 5(')-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which reportedly have anti-angiogenic and anti-cancer effects, were thought to be inhibitors of some types of eukaryotic DNA polymerases. PL moderately inhibited only the activities of calf DNA polymerase alpha (pol alpha), while PN and PM had no inhibitory effects on any of the polymerases tested. On the other hand, PLP, a phosphated form of PL, was potentially a strong inhibitor of pol alpha and epsilon from phylogenetic-wide organisms including mammals, fish, insects, plants, and protists. PLP did not suppress the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as deoxyribonuclease I. For pol alpha and epsilon, PLP acted non-competitively with the DNA template-primer and competitively with the nucleotide substrate. Since PL was converted to PLP in vivo after being incorporated into human cancer cells, the anti-angiogenic and anti-cancer effects caused by PL must have been caused by the inhibition of pol alpha and epsilon activities after conversion to PLP.
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PMID:Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase alpha and epsilon. 1465 74