SUBSTRATE SPECIFICITY OF T5 BACTERIOPHAGE DEOXYRIBONUCLEOSIDE
737
Taking into consideration the capacity of T5 dNMP specific activity of 4 U/mg. T5 dNTP kinase was obtained
kinase to phosphorylate both dNMP and rNMP, we used as described in [10].
the enzyme for the preparative syntheses of [5'-32P]dATP
and [5'-32P]rATP. The synthetic conditions for [5'-32P]dATP
and [5'-32P]rATP from the corresponding [5'-32P]mono-
phosphates are described in the Experimental section.
The two-step phosphorylation, namely, T5 dNMP
kinase-catalyzed phosphorylation followed by a reac-
tion with piruvate kinase and PEP, was performed in
situ without the isolation of intermediate products. We
synthesized [5'-32P]dATP, [5'-32P]dGTP, [5'-32P]dCTP,
and [5'-32P]TTP, as well as [5'-32P]ATP, [5'-32P]GTP
and [5'-32P]CTP, in 80–90% yields and specific activi-
ties of 3–4 kCi/mmol. The attempt to use T5 dNMP kinase
for the preparative phosphorylation of [5'-32P]UMP failed.
A reasonable product yield was not achieved even in
the presence of considerable excesses of the enzyme
after several hours of incubation.
Determination of the Activities of Nucleoside
Monophosphate Kinases
The enzymatic activity of T5 dNMP kinase was
determined using two methods. 1) Spectrophotometri-
cally by the oxidation of NADH. Constants Km and kcat
were determined as described in [9]. The final reaction
mixture 1 ml in volume contained 50 mM Tris-HCl
(pH 7.5), 80 mM KC1, 8 mM MgCl2, 2 mM EDTA,
0.8 mM PEP, 0.2 mM Äíê, 0.1 mM NADH, 2 mM
NMP or dNMP, 20 U lactate dehydrogenase containing
piruvate kinase, and the tested enzyme (0.02–0.2 U).
The reaction was performed in an acryl cuvette at 25°C
for 3–5 min. The rate of NADH oxidation was mea-
sured spectrophotometrically by decreasing the optical
absorption at 340 nm. 1 U was taken as the enzyme
amount capable of catalyzing the conversion of 1 μmol
of dCMP per minute at 25°C. 2) By the phosphoryla-
tion of nucleotides with [γ-32P]ATP as a donor of a
phosphoryl group. The reaction mixture 25 μl in vol-
ume contained a 50 mM Tris–HCl buffer (pH 7.6),
5 mM MgCl2, 1 mM nucleoside 5'-monophosphate,
0.1 M KCl, 0.1 mM ATP, 1 μCi [γ-32P]ATP, and 0.05 U
of the tested enzyme. The mixture was incubated at
37°C. Aliquots (0.5 μl) were taken out in varied time
intervals and loaded on PEI cellulose plates. TLC was
performed in 0.5 M KH2PO4. After the chromatogra-
phy, the plate was dried and the products on PEI cellu-
lose plates were visualized using a Packard Cyclone
Storage Phosphor System.
It is noteworthy that the synthesis catalyzed by T5
dNMP kinase and piruvate kinase was performed in the
presence of a small excess of ATP and a significant
excess of the enzymes and PEP. Such a ratio of the com-
ponents in the reaction mixture is characteristic of the
enzymatic synthesis of phosphorus-labeled com-
pounds. It enables successive NMP phosphorylation by
the common mixing of all reagents in a reaction con-
tainer, a reduction of the reaction time, and the simpli-
fication of the chromatographic purification of the
product. The target product was isolated from the reac-
tion mixture by reverse-phase HPLC in ion-pair mode.
The use of [5'-32P]ATP as a donor of the phosphoryl
group in the phosphorylation of dATP allowed us to
maintain the molar activity of [5'-32P]ATP.
Thus, by using T5 dNMP kinase for the synthesis of
nucleoside 5'-triphosphates labeled with phosphorus-32 at
the α position, we simplified the synthesis with the
retention of high product yields. Apparently, the
obtained results can be used for the synthesis of similar
compounds labeled with phosphorus-33. In general, the
technological approach of the replacement of low-spe-
cific E. coli NMP kinases by a “universal” enzyme capa-
ble of effectively phosphorylating nucleoside 5'-mono-
phosphates is promising and, when properly developed,
can expand into other technological processes where
d/rNMP need phosphorylation.
The activity of T5 dNMP kinase in the reaction with
UMP. The reaction mixture 25 μl in volume contained
a 50-mM Tris–HCl buffer (pH 7.6), 5 mM MgCl2,
0.1 M KCl, 0.1 mM ATP, 300 μCi [5'-32P]ATP, and
0.4 U T5 dNMP kinase. The mixture was incubated at
37°C. Aliquots (0.5 μl) were taken out at 20 and 60 min
and loaded on PEI cellulose plates. TLC was performed
in 0.5 M KCl. The products on PEI cellulose plates
were visualized using a Packard Cyclone Storage Phos-
phor System.
Synthesis of [α-32P]dCTP. A mixture of [5'-32P]dCTP
(10 mCi, 2.5–3 nmol), 5 U T5 dNMP kinase, and 5 U
piruvate kinase was added to the reaction mixture
(100 μl) containing a 50-mM Tris–HCl buffer (pH 8.0),
EXPERIMENTAL
Materials. Tris, phosphoenol piruvate (PEP), NADH 5 mM MgCl2, 0.2 M KCl, 0.05 mM ATP, 5 mM dithio-
(reduced), piruvate kinase, and lactate dehydrogenase con- threitol, and 5 mM PEP. The mixture was incubated at
taining piruvate kinase were from Sigma; plates with PEI 37°C for 30 min. For the determination of the reaction
cellulose were from Merck; [γ-32P]ATP (radioactive con- yield, an aliquot (0.2–0.3 μl) was analyzed by TLC on
centration of 10 mCi/ml, molar activity of 4000 Ci/mmol) a PEI cellulose plate in a 0.5-M potassium phosphate
was purchased from TsKP Phosphor, RussianAcademy of buffer (pH 4.0). The plate was dried and visualized
Sciences. d2CMP was synthesized from 2',3'-dideoxycyti- using a phosphorimager or autoradiography. The prod-
dine and phosphorus oxychloride as described in [13]. All uct yield was 90% relative to radioactivity. The target
[5'-32P]rNMP and [5'-32P]dNMP were prepared as product was isolated by reverse-phase HPLC in ion-
described in [14] with a molar activity of 3000 Ci/mmol. pair mode on a C-18 column in a gradient of ethanol in
E. coli CMP kinase was isolated according to [4] with a 50 mM triethylammonium bicarbonate. The final yield
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009