Catalytic activity of nuclease P1: Experiment and theory Page: 4 of 10
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monophosphates were isolated by reverse phase HPLC. Dinucleoside
monophosphates (6 nmol) were solubilized in 100 gL of reaction buffer (60 mM Na
acetate, pH 5.3, containing 42 gM Zn acetate). Hydrolysis was initiated by addition
of 50 ng of P1 nuclease in 2 gL of buffer. The mixture was incubated at 37* C for the
duration of the reaction which was terminated with the addition of 15 pL of 1 M
TRIS. Decrease of the integrated area of absorption of reactants and increase of the
integrated area of products were monitored by HPLC with detection at UV 254 nm.
Rates of hydrolysis by nuclease P1 for equimolar mixtures of the 16 possible
dinucleoside monophosphates differ by more than two orders of magnitude. Box et
al. (1993) showed that these differences correlated with the 5' nucleoside.
Furthermore, the rate of hydrolysis is reduced if the 5' nucleoside is damaged by
ionizing radiation or oxidative stress, (Box et al. 1993; Weinfeld et al. 1992). Results
of more recent studies shown in Table I support the hypothesis that the 5' nucleoside
is the principal binding locus between enzyme and substrate; however, they indicate
that the 3' nucleoside also influences hydrolytic activity. Substrate d(TFpA), which
d(TpG) d(TpC) d(TpA) d(TpT) d(TpTF)a d(TFpT) d(TGpA)b d(TpGOH)c
112 90 70 40 60 0 3 325
a) TF = Formamido remnant of Thymine
b) TG = R-5,6-Dihydroxy-5,6-Dihydrothymine (Thymine Glycol)
c) GOH = 8-OxoGuanine
Table I: Catalytic Constant (moles substrate hydrolyzed/mole enzyme) for P1
hydrolysis of the phosphodiester bond in dinucleoside monophosphates.
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Miller, J.H.; Falcone, J.M.; Shibata, M. & Box, H.C. Catalytic activity of nuclease P1: Experiment and theory, article, October 1, 1994; Richland, Washington. (digital.library.unt.edu/ark:/67531/metadc666014/m1/4/: accessed February 17, 2019), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.