M. Katane et al.
ArchivesofBiochemistryandBiophysics654(2018)10–18
electrophoresis, proteins in the gel were stained with Coomassie Bril-
liant Blue R-250.
containing 50 mM borate buffer (pH 8.0), 1 mM dithiothreitol and
10 mM 5-oxo-D-Pro in a final volume of 150 μL. The reaction mixture
was incubated at 37 °C for 60 min prior to addition of 600 μL methanol
to stop the reaction. Subsequently, the mixture was incubated at
−80 °C for 1 h and centrifuged at 20,000 × g for 10 min at 4 °C to re-
move precipitated proteins. Supernatant (600 μL) was then filtered
through a 0.45 μm Millex-LH filter before analysis of amino acids by
high-performance LC (HPLC) according to the method of Hashimoto
et al. [44]. Briefly, an aliquot (10 μL) of the filtered solution was mixed
with 30 μL of 400 mM borate buffer (pH 9.0) and 20 μL of OPA/Boc-L-
cysteine reagent (prepared by mixing 10 mg OPA with 10 mg Boc-L-
cysteine in 1 mL methanol) to derivatise amino acids. After incubation
at room temperature for 2 min, a 10 μL aliquot was injected into the
Jasco chromatographic system described above and amino acid deri-
vatives were separated using an octadecylsilyl silica gel column
(Mightysil RP-18GP, 150 × 4.6 mm internal diameter; Kanto Chemical
Co.) with isocratic elution at a flow rate of 1 mL/min. The mobile phase
comprised 50 mM sodium acetate buffer (pH 4.0)/methanol (50:50 v/
v), and spectrophotometric determination was performed at 344 nm.
The negative control reactions in the absence of DGLUCY were also
performed, and the amount of D-Glu produced by the enzymatic reac-
tion was determined by subtracting the peak area in the chromatogram
of the sample in the absence of DGLUCY from the peak area in the
chromatogram of the sample in the presence of DGLUCY.
2.4. Enzyme activity assays
The catalytic activity of DGLUCY was determined by measuring the
formation of 5-oxo-D-Pro from D-Glu as described previously [1] with
some modifications. Specifically, an appropriate amount (100 μg) of
purified enzyme was added to a reaction mixture (at a final con-
centration of ∼10 μM) containing 50 mM borate buffer (pH 8.0), 1 mM
dithiothreitol and 10 mM D-Glu in a final volume of 150 μL. The reac-
tion mixture was incubated at 37 °C for 60 min prior to addition of
600 μL methanol to stop the reaction. The mixture was incubated at
−80 °C for 1 h and centrifuged at 20,000 × g for 10 min at 4 °C to re-
move precipitated proteins. The supernatant (600 μL) was then filtered
through a 0.45 μm Millex-LH filter (Millipore, Bedford, MA, USA), and a
100 μL aliquot of the filtrate was evaporated to dryness. Subsequently, a
65 μL aliquot of 2-propanol was added to the residue to dissolve 5-oxo-
D-Pro but not D-Glu, and the suspension was vigorously vortexed for
20 s, sonicated in a water bath for 5 min and vigorously vortexed for
another 20 s to extract the 5-oxo-D-Pro product. The suspension was
then centrifuged at 20,000 × g for 5 min at 4 °C, and the supernatant
(60 μL) was transferred to a fresh 1.5 mL microtube. The original pellet
was then vigorously vortexed in 65 μL 2-propanol again for 20 s to
further extract the 5-oxo-D-Pro product. The suspension was then cen-
trifuged at 20,000 × g for 5 min at 4 °C and the supernatant (60 μL) was
transferred to a fresh 1.5 mL microtube. The extraction procedure was
repeated one more time and supernatants were combined. The com-
bined 180 μL supernatant was filtered through a 0.45 μm Millex-LH
filter, and an 80 μL aliquot of the filtrate was mixed with 100 μL of
10 mM L-Trp-OMe in acetonitrile/methanol (90:10 v/v) and 20 μL of
50 mM EDC in acetonitrile/methanol (90:10 v/v) to derivatise amino
acids in the mixture (Fig. 1B). After incubation at 65 °C for 1 h, the
solution was filtered through a 0.45 μm Millex-LH filter, and a 5 μL
sample was injected into a Jasco chromatographic system comprising a
model PU-2089 pump, a model UV-2075 UV–visible detector and a
model 807-IT integrator (Jasco Corp., Tokyo, Japan). Amino acid de-
rivatives were then separated on an octadecylsilyl silica gel column
(Mightysil RP-18GP, 150 × 4.6 mm internal diameter; Kanto Chemical
Co., Tokyo, Japan) with isocratic elution at a flow rate of 1 mL/min.
The mobile phase comprised 50 mM sodium acetate buffer (pH 4.0)/
methanol (72:28 v/v), and spectrophotometric determination was
performed at 278 nm. The amount of 5-oxo-D-Pro was determined from
the peak area in the chromatogram. Under these conditions, similar
chromatograms to those reported in our previous study [1] were ob-
tained, and 5-oxo-D-Pro was clearly separated from 5-oxo-L-Pro
(Fig. 1C−E). To verify the formation of 5-oxo-D-Pro from D-Glu, the
fraction corresponding to the 5-oxo-D-Pro peak was isolated and eva-
porated to dryness, and a sample of the residue was dissolved in 50 μL
distilled water and subjected to liquid chromatography-mass spectro-
metry (LC-MS) analysis using a JMS-T100LP time-of-flight mass spec-
trometer (JEOL Ltd, Tokyo, Japan) connected to a 1200 Series LC
system (Agilent Technologies, Santa Clare, CA, USA). An octadecylsilyl
silica gel column (Capcell Core C18, 50 × 2.1 mm internal diameter;
Osaka Soda, Osaka, Japan) was used at a flow rate of 0.4 mL/min, with
a gradient comprising solution A (acetonitrile containing 0.1% [v/v]
formic acid) and solution B (distilled water containing 0.1% [v/v]
formic acid) as follows: 0–8 min, 5%–100% solution A; 8–10 min, 100%
solution A. MS analysis was performed in positive ion mode, and ca-
tionic forms of the derivatised product, [M + H]+ and [M + Na]+ (m/
z = 330.1434 and 352.1269, respectively), were observed in the mass
spectrum (Fig. 1F), confirming the identity of 5-oxo-D-Pro.
To examine the stereospecificity of DGLUCY, L-Glu and 5-oxo-L-Pro
were used as substrates instead of D-Glu and 5-oxo-D-Pro, respectively.
To examine the effects of various compounds on the activity of
DGLUCY, each compound was added separately to the reaction mixture
at a final concentration of 50 μM (FMN, FAD, PL, PLP, NAD+ or
NADP+) or 1 mM (EDTA, MgCl2, CaCl2, MnCl2, CuCl2, ZnCl2, ATP, ADP
or AMP), and relative activity was normalised against that in its ab-
sence. Preliminary experiments showed that none of the compounds
had an effect on the efficiency of extraction or derivatisation of amino
acids. To examine further the effects of MnCl2 on the activity of
DGLUCY, the metal-free form of DGLUCY (prepared as described
above) was used as enzyme. MnCl2 was added to the reaction mixture at
a final concentration of 0.1–1000 μM, and its relative stimulating ac-
tivity was determined by considering the activity of the enzyme in the
presence of 1000 μM MnCl2 as 100%. The 50% effective concentration
(EC50) of this cofactor was determined by fitting data to the following
formula: y = 100 + {–100/[1 + (x/EC50)
slope]}, where y, x, and slope
are the relative activity, concentration of MnCl2, and slope of the curve,
respectively. EC50 value was estimated using the nonlinear least-
squares fitting algorithm in the pro Fit 7.0 software package (Quantum
the effects of pH on the activity of DGLUCY, enzymatic reactions were
performed in 50 mM imidazole-HCl buffer (pH 6.0–8.0) or 50 mM bo-
rate buffer (pH 8.0–10.0) in the presence (D-Glu-to-5-oxo-D-Pro reac-
tion) or absence (5-oxo-D-Pro-to-D-Glu reaction) of 1 mM MnCl2. To
determine the Michaelis constant (Km) and maximal velocity (Vmax
)
values for D-Glu and 5-oxo-D-Pro, D-Glu and 5-oxo-D-Pro (at a final
concentration of 5–80 mM and 10–400 mM, respectively) were used as
substrates and the enzymatic reaction was performed in the presence (D-
Glu-to-5-oxo-D-Pro reaction) or absence (5-oxo-D-Pro-to-D-Glu reaction)
of 1 mM MnCl2 under conditions in which the production of 5-oxo-D-Pro
and D-Glu, respectively, increased linearly with incubation time and
exhibited standard Michaelis-Menten-type properties. Data were fitted
to the Michaelis-Menten equation, and Km and Vmax values were esti-
mated using the nonlinear least-squares fitting algorithm in the pro Fit
7.0 software package. The turnover number (kcat) was calculated from
Vmax and the estimated molecular mass of the recombinant protein
(65,874 Da for N-terminally His-tagged mouse DGLUCY).
The catalytic activity of DGLUCY was also determined by measuring
the formation of D-Glu from 5-oxo-D-Pro and H2O as described pre-
viously [1] with some modifications. Specifically, an appropriate
amount (100 μg) of purified enzyme was added to a reaction mixture
2.5. Absorbance spectrum of DGLUCY
Absorbance spectra of mouse DGLUCY were measured using a plate
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