Figure 3. Mammalian DNA polymerase inhibition dose-response curves of PLP. The enzymes used
triangle), human pol δ (closed reverse-triangle), human pol ε (closed circle) and human pol λ (open
diamond). Pol activity in the absence of the compound was taken to be 100 %. Data are shown as the
means ± SEM of three independent experiments.
inhibitory effect of PLP on topos I and II activities was as strong as that on pols γ and δ
activities. On the other hand, no vitamin B6 compounds could inhibit the activities of
prokaryotic pols such as the Klenow fragment of E. coli pol I, T4 pol and Taq pol, and the
other DNA-metabolic enzymes such as calf terminal deoxynucleotidyl transferase, HIV
reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I (Table 1). These
results suggested that PLP was the strongest inhibitor of eukaryotic pols and human topos in
the vitamin B6 compounds tested, and PLP could strongly inhibit replicative pols α and ε in
the DNA metabolic enzymes tested.
Since most PL is thought to be converted to PLP in vivo (see the latter part of this
review), the anti-angiogenic and anti-cancer effects of PL may be caused by converted PLP
in the cells; therefore, PLP may be a key agent for analyzing the in vivo functions of
replicative pols. The remainder of this review is thus devoted to an analysis of PLP inhibition
of pols and in vivo cell conversion from PL to PLP.
Inhibition of DNA Polymerase and Topoisomerase by Vitamin B6 11
Table 2. Effects of poly (rC), bovine serum albumin (BSA) or Nonidet P-40 (NP-40) on
the inhibition of DNA polymerase activities by PLP
Compounds added to the reaction mixture Calf DNA polymerase α (%)
100 μM PLP + 100 μg/ml poly (rC) 1.5
100 μM PLP + 100 μg/ml BSA 2.2
Compounds added to the reaction mixture Human DNA polymerase ε (%)
100 μM PLP + 100 μg/ml poly (rC) 1.1
100 μM PLP + 100 μg/ml BSA 0.7
100 μM poly (rC) and 100 μg/ml BSA or 0.1 % NP-40 was added to the reaction mixture.
In the absence of PLP, DNA polymerase activity was taken as 100 %.
3. EFFECTS OF REACTION CONDITIONS ON DNA
To determine the effects of a non-ionic detergent on the binding of PLP to replicative
pols, a neutral detergent, Nonidet P-40 (NP-40), was added to the reaction mixture at a
concentration of 0.1 %. In the absence of PLP, pol activity was taken as 100 %. The pols α or
ε inhibitory effect of PLP at 100 μM was not affected by the addition of NP-40 to the reaction
mixture (Table 2), implying that the binding interaction to the enzyme by PLP is hydrophilic.
We also tested whether an excess amount of a substrate analogue, poly(rC) (100 μg/ml), or a
protein, BSA (100 μg/ml), could prevent the inhibitory effects of PLP to determine whether
the effects of the compound were due to their non-specific adhesion to the enzymes, or to
12 Yoshiyuki Mizushina, Norihisa Kato, Hiromi Yoshida et al.
selective binding to specific sites. Poly(rC) and BSA had little or no influence on the effects
of PLP, suggesting that binding to pols occurs selectively (Table 2).
Next, to elucidate the inhibition mechanism of PLP, the extent of inhibition as a function
of the DNA template-primer or nucleotide substrate concentrations was studied. Table 3
shows the result of our kinetic analysis of PLP. In kinetic analysis, poly (dA)/oligo(dT)12-18
and dTTP were used as the DNA template-primer and nucleotide substrate, respectively.
Table 3. Kinetic analysis of the inhibitory effects of PLP on the activities of mammalian
DNA polymerases α and ε, as a function of the DNA template-primer dose and the
nucleotide substrate concentration
Enzyme Substrate PLP (μM) Kma) (μM) Vmaxa)
a) These data were obtained from Lineweaver Burk plot;
b) These data were obtained from Dixon plot;
c) i.e., poly(dA) / oligo(dT)12-18;
Double reciprocal plots of the results show that the PLP inhibition of pol α activity did
not compete with the DNA template-primer and acted by competing with the nucleotide
substrate. In the case of the DNA template-primer, the apparent Michaelis constant (Km) was
unchanged at 13.0 μM, whereas 73.2 % decreases in maximum velocity (Vmax) were
observed in the presence of 30 μM PLP. The Vmax for the nucleotide substrate (dTTP) was
Inhibition of DNA Polymerase and Topoisomerase by Vitamin B6 13
unchanged at 29.2 pmol/h, and the Km for the nucleotide substrate increased from 1.65 to
9.09 pmol/ml in the presence of zero to 30 μM PLP. The inhibition constant (Ki) value,
obtained from Dixon plots, was found to be 18.3 μM and 11.3 μM for template-primer DNA
and nucleotide substrate dTTP, respectively.
Similarly, the inhibition of pol ε by PLP did not compete with the DNA template-primer
but competed with the nucleotide substrate since there was no change in the apparent Km
(6.25 μM) for the DNA template-primer, while the Vmax for the DNA template-primer
decreased from 37.0 to 10.0 pmol/h DNA template in the presence of zero to 30 μM PLP. On
the other hand, the apparent Vmax for the nucleotide substrate was unchanged at 58.8
pmol/h, whereas a 4.25-fold increase in the Km was observed in the presence of 30 μM PLP.
The Ki value was 16.5 μM for the DNA template and 6.4 μM for the substrate dTTP. The
inhibitory mode of pol α by PLP was found to be the same mode as pol ε.
In pols α and ε, the Ki value for the DNA template-primer was higher than that for the
nucleotide substrate, suggesting that the affinity of PLP to pols α and ε is higher at the
nucleotide substrate-binding site than at the DNA template-binding site. Since PLP bears a
structural resemblance to both the DNA template-primer and the nucleotide substrate, the
structures of pol α and pol ε may incorporate the PLP molecule more acceptably than
authentic nucleotides. At least, as far as pols α and ε are concerned, PLP binds to the
enzymatic active region when competing with the nucleotide substrate, and subsequently
When activated DNA and four deoxyribonucleoside triphosphates (dNTPs) were used as
the DNA template-primer and nucleotide substrate, respectively, the inhibition of pols α and ε
by PLP was the same as when using poly(dA)/oligo(dT)12-18 and dTTP (data not shown).
The mode of inhibition was suggested to be ineffective for pyrimidine deoxyribonucleoside
triphosphates (dCTP and dTTP) and purine deoxyribonucleoside triphosphates (dATP and
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