1
Characterization of NAD+-Analogues using a High Pressure Liquid
Chromatography-based NAD+-Glycohydrolase Assay
Susan P. Yates, and A. Rod Merrill*
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G
2W1, Canada.
*
Corresponding author.
Tel: (519) 824-4120 ext. 53806.
Fax: (519) 837-1802.
E-mail:
rmerrill@uoguelph.ca
Subject Category: Enzymatic Assays and Analyses
1Abbreviations:
ADPR, ADP-ribose; ADPRT, ADP-ribosyltransferase; CS, chelating
sepharose; DT, Diphtheria toxin; eEF2, eukaryotic elongation factor 2; ETA, exotoxin A; FNAD+,
2’-F-ribo-NAD+;
GH,
NAD+-glycohydrolase;
HPLC,
high
pressure
liquid
chromatography; IC50, inhibitory concentration for 50% inhibition of enzyme activity; kcat,
catalytic rate constant; KD, substrate binding (dissociation) constant; N-Ac-L-Phe, N-acetyl-Lphenylalanine;
-NAD+,
1,N6-etheno-NAD+;
PABA,
4-aminobenozic
acid;
PE24H,
Pseudomonas aeruginosa exotoxin A 24 kDa C-terminal fragment containing a hexa-Histag;
Vmax, maximal velocity
2
Abstract (200 words)
A high pressure liquid chromatography (HPLC)-based technique was developed to assess
the NAD+-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A
containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the
substrate (NAD+) and products (ADP-ribose and nicotinamide) produced over the reaction time
course, whereby the peak area of nicotinamide is correlated using a standard curve. This
technique was used to determine whether the NAD+-analogue, 2’F-ribo-NAD+, was a competing
substrate or a competitive inhibitor for this toxin. This NAD+-analogue was hydrolyzed at a rate
of 0.2% that of NAD+, yet retained the same binding affinity for the toxin as the parent
compound. Finally, the rate that a fluorescent NAD+-analogue, -NAD+, is hydrolyzed by the
toxin was investigated. This analogue was hydrolyzed 8 times slower compared to NAD+ as
determined using HPLC. The rate of hydrolysis of -NAD+ calculated using the fluorometric
version of the assay shows a 6-fold increase in reaction rate compared to that determined by
HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses
NAD+-glycohydrolase activity and offers the advantage of directly measuring the enzymecatalyzed hydrolytic rate of NAD+ and its analogues.
Keywords
HPLC,
mono-ADP-ribosyltransferase,
hydrolysis, NADase
NAD+-analogues,
NAD+-glycohydrolase,
NAD+
3
Introductory Statement
The ubiquitous NAD+ glycohydrolases, also termed NADases (EC 3.2.2.5), have been
described for a wide range of prokaryotic and eukaryotic systems [1,2]. Generally, prokaryotes
possess soluble NADases whereas eukaryotes possess membrane-associated NADases [2].
These enzymes catalyze the hydrolysis of the nicotinamide-ribose bond of the -anomer of
NAD+, with the transfer of ADP-ribose to water and the release of nicotinamide (Fig. 1A) [1,2].
These enzymes were first identified in 1942 [3], however, the exact physiological function still
remains unclear [2]. New evidence is emerging that several NADases also catalyze the synthesis
and hydrolysis of cyclic ADP-ribose, thereby serving as a second messenger in the regulation of
intracellular calcium levels [4].
The mono-ADP-ribosyltransferases, which include several bacterial toxins, also cleave
the nicotinamide-ribosyl bond, however, preferentially transfer the ADP-ribose moiety to a
protein acceptor.
The bacterial toxins, Pseudomonas aeruginosa exotoxin A (ETA) and
diphtheria toxin (DT), use eukaryotic elongation factor 2 (eEF2) as the protein acceptor. Cholera
toxin ADP-ribosylates an arginine in the -subunit of guanine nucleotide-binding protein and
pertussis toxin modifies a cysteine in the G family of proteins (Gi, Go, Gt) (new ref#1).
Chlostridium botulinum C3 exotoxin specifically ribosylates the RhoA, B and C isoforms of the
Rho and Rac families at Asn residues (new ref#2).
Actin is the target substrate for C.
perfringens iota toxin, which modifies an arginine residue (new ref#3). However, in the absence
of the appropriate protein substrate, these toxins possess a slow NAD+-glycohydrolase activity
wherein NAD+ is hydrolyzed to nicotinamide and ADP-ribose [5] as is the case for the classic
NADases. No physiological role has been assigned to this hydrolysis reaction for these toxins
[6-8]. However, NAD+ is hydrolyzed by ETA at a rate that is 25,000-55,000 times slower
4
compared to the rate that it transfers ADP-ribose to eEF2 [9-11] and DT shows 1700-80,000 fold
slower hydrolysis of NAD+ in the absence of eEF2 [7,12]. Since the hydrolysis of NAD+
(NADase activity) occurs at a fraction of the ADP-ribosyltransferase rate, the former reaction is
not believed to be biologically significant [7].
However,
measurement
of
NAD+-glycohydrolase
activity
for
mono-ADP
ribosyltransferases like ETA is useful for assessing the integrity of the NAD+-binding site and
for indirectly measuring the affinity that NAD+ (or NAD+ analogues) have for the active site of
wild-type and mutant toxin proteins [9]. It is also an essential tool for probing the underlying
reaction mechanism(s) for the ribosyltransferase enzymes.
The NAD+-glycohydrolase activity from Neurospora crassa Conidia, an NADase, was
previously characterized using reverse-phase HPLC [13].
The study herein reports the
development of an HPLC-based NAD+-glycohydrolase assay specific to the catalytic domain of
ETA (and other ADP-ribosyltransferases) containing a hexa-His tag (PE24H) as adapted from
the previous technique [13]. Competing substrates and competitive inhibitors were distinguished
using this assay through a study of an NAD+ analogue, 2’-F-ribo-NAD+ (F-NAD+). F-NAD+
contains a fluorine substitution at the 2’-OH position of the nicotinamide-ribose (Fig. 1B). In
addition, the rate of -NAD+ hydrolysis by PE24H was quantified and compared using both the
HPLC- and fluorescence-based [11] approaches. -NAD+ is the compound employed in the
fluorometric NAD+-glycohydrolase assay (Fig. 1B) [10,11].
5
Materials and Methods
Overexpression and purification of PE24H
The catalytic fragment of ETA with a C-terminal 6-His tag (PE24H) was overexpressed
and purified as described [11]
IC50 determination for F-NAD+
In the fluorescence-based ADPRT assay, the substrates, -NAD+ and eEF2 (purified as
described earlier [11]), were used at saturating doses of 500 M and 14 M, respectively, and a
range of F-NAD+ concentrations were added to the assay medium (20 mM Tris-HCl, pH 7.9) in a
70 L total volume. The reaction mixture was incubated for 5 min at 25C in a disposable
UltraVette™ microcuvette (Brand GMBH Co., Wertheim, Germany). The reaction was initiated
by adding 5 nM (final concentration) PE24H. The concentration of the inhibitor that reduces the
activity of the enzyme by 50%, the IC50 value, was determined by non-linear regression curve
fitting using Origin 6.1 (OriginLab, Northhampton, MA). The data were fit to the exponential
first-order decay function.
Fluorescence-based NAD+-glycohydrolase (GH) assay
To determine if the mobile phase inhibited the GH activity of PE24H, the hydrolysis of
500 M -NAD+ by 1 M wild-type PE24H was measured in either buffer (20 mM Tris-HCl, pH
7.9), mobile phase (20 mM NaHPO4, pH 5.5: acetonitrile (100:5 v/v %)), or mobile phase with
internal standard (PABA at 2.5 g/mL) in an ultramicrocuvette (3-mm path length; Helma Inc.,
Concord, ON, Canada) using a Varian Cary Eclipse fluorometer (Varian Canada, Inc.;
Mississauga, ON). An excitation wavelength of 305 nm and an emission wavelength of 410 nm
6
were used with band passes fixed at 5 nm and the gain set to 600 V. The reaction was monitored
for 4 hours at 25C. A control containing -NAD+ in buffer only (without toxin) was also
measured. The reaction mixtures containing mobile phase (with and without internal standard)
were 75% mobile phase and 25% buffer. The rate of reaction was determined using the linear-fit
function in the Origin 6.1 software package.
In order to determine the rate of hydrolysis of -NAD+ under the same conditions as the
HPLC-based technique, the ultramicrocuvette contained 450 M -NAD+ and 10 M wild-type
PE24H in buffer. The reaction was monitored for 4 hours at 25C using the Varian Cary Eclipse
fluorometer (excitation and emission set to 305 and 410 nm, respectively). Both band passes
were at 5 nm with the gain at 500 V. Inclusion of -NAD+ in the absence of toxin was used to
correct for hydroxide ion-catalyzed hydrolysis of the substrate.
A standard curve was generated using the fluorescence emission of -AMP at
concentrations ranging from 0 to 12 M in buffer. This standard curve was used to calibrate the
reaction rate.
HPLC instrumentation
Samples and standards were injected into the HPLC using a 10 L sample loop. The
HPLC system consisted of a Bio-Rad HRLC Model 2700 (Mississauga, ON, Canada) with sample
detection by a Linear UV-205 absorption monitor set at 259 nm. A Micro-Guard® cartridge (30 x
4.6 mm; Bio-Rad) was used in-line and was followed by a RoSiL C18 HL column (150 mm x 4.6
mm, 3 M particle size; Bio-Rad). The mobile phase (20 mM NaHPO4, pH 5.5: acetonitrile
(100:5 v/v %)) was pumped through the system with a flow rate of 0.8 mL/min at 12 MPa.
7
Nicotinamide standard curve
A nicotinamide standard curve was prepared where nicotinamide (0-500 pmoles) in 25
L buffer (20 mM Tris-HCl, pH 7.9) was added to 75 L mobile phase containing an internal
standard (2.5 g/mL 4-aminobenzoic acid (PABA) or 300 g/mL N-acetyl-L-phenylalanine (NAc-L-Phe)) and was injected into the HPLC system. The area of the nicotinamide product peak
was determined using the baseline tool in Origin 6.1 and calibrated with the chosen internal
standard. The nicotinamide standard curve correlates the total nicotinamide peak area to the
number of moles (or concentration) of nicotinamide produced during the reaction time.
Base hydrolysis of NAD+
NAD+ (500 M) was prepared in 500 L water. A 20 L aliquot was taken and 80 L
mobile phase containing an internal standard (PABA; 2.5 g/mL) was added (t = 0). The sample
was injected into the HPLC as described above. NaOH (40 mM, final concentration) was added
to the remaining sample and aliquots (20 L) were taken at 1, 2, 3, 4, 5 min and prepared for
HPLC injection as was described for the initial sample.
The nicotinamide peak area was
correlated to the number of pmoles of nicotinamide produced using the nicotinamide standard
curve.
Affinity chromatography
Chelating Sepharose (CS) Fast Flow (100 L; Amersham Biosciences, Baie d’Urfé,
Canada) was prepared in a Bio-Rad spin column (previously charged with 100 mM ZnSO4 and
equilibrated with mobile phase).
8
In a total volume of 200 L, 500 pmoles of nicotinamide (or NAD+) in 25:75 (v/v %)
water: mobile phase (with internal standard) were prepared. To the spin-column, 100 L of the
sample was loaded and the flow-through collected (after spinning). HPLC was performed using
a 10 L sample loop on both samples ((i) sample not loaded to the CS column and (ii) sample
from CS column flow-through). The peak areas (corrected using the internal standard) of
nicotinamide (or NAD+) were then compared.
PE24H (10 M) in mobile phase (with internal standard) was prepared in a final volume
of 200 L. To the CS spin-column, 100 L of the sample was loaded and the flow-through
collected (after spinning). A BCA assay (Pierce Biotechnology, Rockford, IL, U.S.A.) was used
to compare protein levels from the sample not loaded to the CS column and the sample from the
CS column flow-through.
HPLC-based NAD+-glycohydrolase (GH) assay
Reactions were performed at 425 M NAD+ in the presence of PE24H (10 M final
concentration) at 25C for up to 4 h, in a final volume of 265 L in 20 mM Tris-HCl, pH 7.9.
Aliquots (25 L) were removed at t = 0, 1, 2, 3, 4 hours followed by the addition of 75 L of
mobile phase containing internal standard (2.5 g/mL PABA or 300 g/mL N-Ac-L-Phe). Each
sample mixture was then loaded to a separate Bio-Rad spin column containing 100 L of CS
resin (previously charged with 100 mM ZnSO4 and equilibrated with mobile phase) to remove
PE24H (via interaction with the His-tag). The flow-through (after spinning) was collected and
injected into the HPLC.
When evaluating F-NAD+, the toxin concentration was increased to 47.9 M and the
reaction was monitored for 48 hours. Due to the slow hydrolysis rate, the nicotinamide peak
9
could not be monitored, therefore the ADP-ribose (ADPR) peak was observed, however the
ADPR peak overlaps with F-NAD+ (similarly in the case of NAD+), therefore, the peaks were
mathematically deconvoluted to determine the area of ADPR alone. Since a standard curve to
correlate the peak area of ADPR to the number of moles of ADPR was not available, the same
analysis on the ADPR peaks produced from NAD+ (toxin at 10 M) was performed in parallel.
Comparing the peak areas for ADPR (calibrated to the internal standard) from both F-NAD+ and
NAD+ over time and accounting for differences in toxin concentration, the rate of hydrolysis was
determined.
The hydrolysis of -NAD+ (450 M) was determined as described above for NAD+
however, this measurement required the use of the internal standard, N-Ac-L-Phe, since -NAD+
and PABA possessed the same retention time.
Binding affinity of F-NAD+ to PE24H
The quenching of the intrinsic tryptophan fluorescence of PE24H was used to determine
the binding constant (KD) for F-NAD+ as described elsewhere [14]. Briefly, the fluorescence
quenching of PE24H was measured as a function of F-NAD+ concentration. Triplicate reactions
were performed over a suitable concentration range of F-NAD+, in the presence of PE24H (1.25
M, final concentration) at 25C in an initial volume of 600 L (50 mM NaCl, 20 mM Tris-HCl
(pH 7.9) buffer). Samples were excited at 295 nm and monitored at an emission of 340 nm. All
band passes were set to 4 nm and all measurements were corrected for buffer contribution to the
fluorescence signal.
10
Results
Development of the HPLC-based NAD+-glycohydrolase (GH) assay
Initially, each component (e.g. NAD+, nicotinamide, PABA) was injected into the HPLC
system individually in order to identify the retention times associated with the various
compounds on the chromatogram (data not shown).
The peaks on the chromatograms were then calibrated with an internal standard. A range
of concentrations of the standard, PABA (0.1 – 50 g/mL), was injected into the HPLC along
with nicotinamide (200 – 500 pmoles).
The internal standard peak was compared to the
nicotinamide peak and an appropriate concentration of the internal standard (PABA at 2.5
g/mL) was chosen for use in the assay.
Known amounts of nicotinamide were prepared in mobile phase containing PABA (2.5
g/mL).
The internal standard was used to standardize the peak area associated with
nicotinamide in order to generate a standard curve (Fig. 2A).
Base hydrolysis of NAD+ was first performed to determine whether this HPLC technique
would be sensitive enough to measure a reaction rate. Fig. 3 shows both the chromatogram
illustrating the appearance of products and the reaction coordinate. The reaction rate is 50
pmoles of nicotinamide product per minute.
When measuring the PE24H GH activity, all protein must be removed before loading
samples to the reverse-phase column. We took advantage of the His-tag on PE24H to remove it
from the reaction mixture. A series of experiments were performed to ensure this approach was
suitable.
First, small spin columns containing chelating sepharose (CS) were charged with ZnSO4
and equilibrated with mobile phase. Initially the reaction mixture was passed through the
11
column to determine whether the non-protein reaction components would be lost. Samples of
known amounts of nicotinamide (or NAD+) in mobile phase containing the internal standard
were prepared. From the sample, 100 L was removed and injected into the HPLC (before CS
column sample). The remaining 100 L was loaded to the CS column and the eluent injected
into the HPLC (after CS column sample). Although some nicotinamide (or NAD+) was lost
using the column, the same proportion of the internal standard was also lost. Therefore, the peak
area ratios of nicotinamide and NAD+ to the internal standard remained the same with or without
CS column chromatography. However, there was a decrease in assay sensitivity based on the
diminished peak areas.
Next, it was evaluated whether PE24H could bind to the CS column resin in the presence
of mobile phase. The toxin protein was prepared in mobile phase (with internal standard), and
only half of the protein sample was loaded onto and eluted from the column (after CS column
sample). Both the pre- (not applied to the CS column) and the post-column samples were
analyzed using a BCA assay to determine the amount of protein lost by CS chromatography.
Comparison of the absorbance values showed that near complete binding of PE24H to the CS
column occurred since the post-CS column sample had the same absorbance as the control
(mobile phase with internal standard only) whereas the pre-CS column sample had significantly
higher absorbance.
When measuring GH acitivity, each time point sample required the reaction to be stopped
through inhibition of the toxin. To test whether the mobile phase itself inhibited the reaction, the
fluorometric GH assay was then employed. As seen in Fig. 4, the reaction rate was similar in the
presence of mobile phase to the control (no toxin) reaction. Therefore, the mobile phase was
used to halt the reaction when time course samples were taken.
12
Finally, the GH activity of PE24H was measured using the HPLC approach. Initially
several reaction conditions were investigated, which included toxin concentration, total reaction
volume and sample size, to establish a compromise between sensitivity without requiring large
amounts of substrate or enzyme. The general protocol requires 10 M PE24H in a 250 L
reaction volume, with samples (25 L) taken every hour. Mobile phase (containing internal
standard; 75 L) was added to inhibit further reaction. The total sample (100 L) was applied to
the column and then eluted. The eluent (10 L) was then injected onto the reverse-phase HPLC
column and the peak area for the nicotinamide product was calibrated against the internal
standard peak area. This peak area was correlated to the amount of nicotinamide product using
the nicotinamide standard curve. The hydrolysis of the substrate NAD+ (450 M) by the toxin
(10 M) showed that the production of nicotinamide was linear for 4 hours with a rate of 64 (
7) M nicotinamide produced per hour (Fig. 5A). This NAD+ concentration is near or at the
Vmax; therefore, the kcat was estimated to be 6.4 h-1 in agreement with previously reported values
[10]. Representative chromatograms for NAD+ hydrolysis are shown in Fig. 5B,C.
Characterization of F-NAD+
The IC50 for F-NAD+ was determined to be 82.4  7.4 M (Fig. 6A). However, it was
not clear whether F-NAD+ was acting as a competing substrate or a competitive inhibitor since it
does possess the nicotinamide-ribosyl bond targeted by the toxin. Unfortunately, the ADPRT
fluorometric assay [11] cannot distinguish between these possibilities since the fluorescent label
is part of the NAD+ substrate only.
Therefore, a direct measure of product formation was
devised, whereby the ability of the toxin to hydrolyse NAD+ (or NAD+-analogues) in the absence
of eEF2 (GH activity) was measured using the HPLC-based technique.
13
When evaluating F-NAD+, under the same experimental conditions as for NAD+ (Fig. 5),
no production of nicotinamide was evident even after 18 hours (the chromatograms at 0 and 18
hours are superimposable (data not shown)). Therefore, the toxin concentration was increased
approximately 5-fold and the reaction monitored for 48 hours (Fig. 7A,B). Analysis of the
chromatograms suggested that no or negligible amounts of nicotinamide were produced,
however, some F-NAD+ was hydrolysed since the area for the ADP-ribose (ADPR) peak (the
other reaction product) increased with time (Fig. 7A,B). The extinction coefficient for ADPR is
greater than for nicotinamide; therefore, it would be more sensitive to monitor the appearance of
ADPR than the other product, nicotinamide. In the absence of a standard curve to correlate the
peak area of ADPR with the number of moles of ADPR, the same analysis was performed on the
ADPR product peaks from both NAD+ and F-NAD+ substrates. Comparing the increase in the
peak areas for ADPR (calibrated to an internal standard) from these compounds over time, it was
estimated that the hydrolysis of F-NAD+ is 570 ( 40) times slower than NAD+ (or 0.2% that for
NAD+) (Fig. 7C,D). The chromatogram of F-NAD+ in the absence of toxin (Fig. 7A) possesses
small peaks that correspond to ADPR and nicotinamide reaction products indicating that small
amounts of hydrolysis products were present in the sample.
This amount of ADPR and
nicotinamide was taken to represent the baseline level. In addition, in the absence of toxin over
the same time course (0 to 48 hours) the increase in the ADPR peak produced from F-NAD+
hydrolysis was negligible (data not shown). Therefore, the increase in the ADPR peak is due to
a toxin-catalyzed event and not the breakdown of F-NAD+. Furthermore, the binding of F-NAD+
to PE24H indicated that this fluoronucleotide exhibits a similar affinity for the active site of the
toxin as the NAD+ substrate (Fig. 6B). The dissociation constants (KD) for F-NAD+ and NAD+
are 33 ( 1) and 53 ( 2) M, respectively.
14
Characterization of -NAD+
In our fluorometric assay the NAD+ analogue, -NAD+ is used as a surrogate substrate
[11]. With a direct measure of GH activity now available, it was asked whether the rate at which
-NAD+ is cleaved is comparable to NAD+. Unfortunately, -NAD+ has a similar retention time
as the internal standard, PABA; therefore, a new internal standard was required in order to test NAD+ using the HPLC approach. The compound, N-acetyl-L-phenylalanine (N-Ac-L-Phe) was
chosen as the new internal standard, since its possessed a longer retention time that the other
reaction components. Unfortunately, the column run time had to be increased from 10 to 15
minutes. A range of concentrations of N-Ac-L-Phe (2.5 – 1000 g/mL) was evaluated and 300
g/mL was the chosen concentration to use in the mobile phase. A nicotinamide standard curve
was calibrated using N-Ac-L-Phe (Fig. 2B) at this concentration.
As with the standard PABA, the same preliminary studies involving the CS column were
repeated (as described above).
Like the internal standard PABA, the peak area ratios of
nicotinamide to the new internal standard remained the same for those samples before and after
the column step, therefore the reaction components were lost by the same percentage.
The rate of hydrolysis of -NAD+ versus NAD+ was then assessed by HPLC (using the
internal standard N-Ac-L-Phe). The rate of hydrolysis for NAD+ determined with the new
internal standard was similar to the rate shown in Fig. 5A.
The chromatograms for the
hydrolysis of -NAD+ by PE24H are shown in Fig. 8A,B. The rate of hydrolysis of -NAD+ was
8.6 ( 0.3) M nicotinamide produced per hour (or a kcat of approximately 0.86 h-1) (Fig. 8C).
Therefore, it was concluded that -NAD+ is a poorer substrate for toxin GH activity than NAD+
since it was hydrolysed nearly 8 times slower than natural substrate.
15
The rates of hydrolysis for -NAD+ were determined using the HPLC- and fluorescencebased methods. The rate using the fluorescence-based approach was 50 ( 4) M -ADPR
produced per hour (or a kcat of approximately 5.0 h-1) (Fig. 8D). The fluorescence intensity is
standardized using known amounts of the fluorescence compound -AMP, which is to represent
the -ADPR product. Since each hydrolyzed molecule of NAD+ is cleaved into ADPR and
nicotinamide, the amount of ADPR produced will also equal the amount of nicotinamide
produced.
Therefore the rate determined using the fluorescence-based approach is
approximately 6 times faster than the direct measure HPLC technique.
Discussion
NAD+-glycohydrolase (GH) activity of PE24H can be measured using a fluorometric
assay with -NAD+. The analogue, -NAD+, differs from NAD+ by the addition of an etheno
bridge between the 1 and N6 positions on the adenine ring (Fig. 1) which gives rise to its
fluorescence. However, the intrinsic fluorescence of -NAD+ is quenched intramolecularly by
the nicotinamide ring portion of the molecule. In the hydrolysis reaction the N-glycosidic bond
is cleaved releasing nicotinamide, the intramolecular quencher, causing the fluorescence from
the etheno bridge to increase [15]. This increase in fluorescence can be measured as a function
of time. The reaction rate is calibrated by measuring the fluorescence emission at different
concentrations of -AMP, which represents the fluorescent product of -NAD+ hydrolysis, ADPR. This -AMP standard curve converts the fluorescent intensity seen in the enzymecatalyzed reaction into units of concentration. This fluorometric assay has the advantage that it
is continuous, fast, and simple to use; however, the disadvantage is that it can only evaluate
compounds, specifically NAD+ analogues, which contain the etheno bridge.
16
The compound F-NAD+ was previously shown to be an extremely slow substrate for
CD38 (a classic NADase) and other enzymes that cleave the nicotinamide-ribosyl bond (N.J.
Oppenheimer, personal communication). This study addressed whether the same was true for
ADP-ribosyltransferase toxins like ETA.
Since F-NAD+ lacks the necessary fluorescence
properties exploited in -NAD+, the fluorometric assay was not possible. Therefore, rather than
synthesizing F-NAD+ containing the required etheno bridge, a technique was developed to
measure GH activity of the toxin directly; thus establishing an HPLC-based assay.
Using HPLC the amount of the product, nicotinamide, is quantified directly by
correlating the peak area from the chromatogram to the number of pmoles. Each sample is
calibrated using an internal standard. Two internal standards are available for use. The internal
standard, PABA was originally chosen since it was used in the HPLC approach to which the one
presented herein is based [13]. However, N-Ac-L-Phe was added to the repertoire of standards
available in instances where conflicting retention times between PABA and the reaction
components exist. This was seen in the case of -NAD+. Since N-Ac-L-Phe resolves later than
the reaction components on the chromatogram (Fig. 8A,B), it is recommended that in future
studies this be the chosen standard. The peak for PABA is quite close to the peak for NAD+
(Fig. 5B,C) and the potential exists for overlap to occur if the resolution of the chromatogram is
not adequate.
The protein, PE24H, is removed from the time course samples through inclusion of
chelating sepharose, which binds the His-tag of PE24H. The disadvantage of this step in the
protocol is the decrease in sensitivity. Fortunately, the peak ratios between substrate/product and
the internal standard remain the same.
17
The nicotinamide peak area is examined over time to establish the enzymatic rate.
Nicotinamide is monitored since its peak does not conflict with any of the reaction components
(Fig. 5C); however, because its extinction coefficient is low, assay sensitivity decreases. The
other product ADPR overlaps with the NAD+ peak. At the time this assay was developed,
ADPR was not available to generate the standard curve; in addition, the requirement of
deconvoluting the ADPR and NAD+ peaks was seen as a disadvantage. However, as discussed
in the case of F-NAD+, when slow hydrolysis occurs and the increase in the peak area for
nicotinamide is negligible, the ADPR peak is utilized (Fig. 7).
Based on the GH assay data, F-NAD+ was cleaved at 0.2% the rate that NAD+ was
hydrolyzed by PE24H (Fig. 7). Nevertheless, F-NAD+ exhibits the same level of binding affinity
to the toxin as NAD+ (Fig. 6B). Therefore, F-NAD+ binds to the enzyme but cannot be readily
hydrolyzed. The only structural difference between NAD+ and this analogue is a fluorine
substitution at the 2’-OH position of the nicotinamide-ribose (Fig. 1). In the structure of -TAD
in complex with the catalytic domain of ETA, a hydrogen bond was observed to occur between
Glu-553 and the 2’-OH of the thiazole- (analogous to the nicotinamide) ribose [16]. This
hydrogen bond has been implicated in maintaining NAD+ in a sterically strained conformation
that facilitates the breaking of the nicotinamide-ribosyl bond. The loss of this important H-bond
interaction may account for a reduction in the ability of F-NAD+ to be hydrolyzed by the
enzyme. However, since it possesses many of the same interactions within the active site as
NAD+, F-NAD+ is able to bind with the same affinity. Although F-NAD+ is not a potent
inhibitor, its slow rate of hydrolysis by the toxin makes this compound a useful NAD+-analogue.
This analogue co-crystallized with PE24H would provide important information on the chemical
18
and structural requirements for NAD+ substrate binding to the active site as compared with TAD, since the structure of F-NAD+ is more akin to the natural substrate.
The fluorescent compound, -NAD+ was also assessed using the HPLC NAD+glycohydrolase assay. The rate of hydrolysis of -NAD+ determined using HPLC was 8 times
slower than for NAD+ (Fig. 5A, 8C). This may be a reflection of the addition of the etheno
bridge causing subtle differences that affect the ability of the toxin to cleave the bond. Minor
alterations in the positioning of the NAD+ analogue within the active site or modified properties
for the nicotinamide-ribosyl bond may be the cause. However, the affect is only a factor of 8,
which is significant but is not severely detrimental. In the fluorometric assay, where -NAD+ is
used, the rate to which -NAD+ is hydrolyzed by the wild-type enzyme serves as the reference
for comparisons.
The rate that -NAD+ is hydrolyzed using the fluorescence approach is 6 times faster than
that determined using HPLC (Fig. 8C,D). Many factors must be considered to rationalize this
difference. The fluorescence technique is more sensitive since it continually measures the
increase in fluorescence.
However, to correlate the fluorescence signal it uses an -AMP
standard curve. -AMP represents the closest available compound to the product -ADPR.
However, lacking the ribose and an additional phosphate may change its quantum yield enough
to introduce error, thereby, the rate determined is an overestimation of the true rate. Another
consideration is that the HPLC-based technique is less sensitive since it is a discontinuous assay,
thus the rate is determined from fewer data points, and the nicotinamide peak is comparably
smaller to the other reaction components.
Therefore, the experimental rate may be an
underestimation of the actual rate. However, the chromatograms allow for visualization of
quantities as substrate goes to products. Nevertheless, although the HPLC technique is less
19
sensitive, it directly measures product production whereas the fluorometic assay is indirect since
it relies on a fluorescence change, which is assumed to relate to bond cleavage. Therefore, the
rates determined by these two techniques are similar given the limitations of each approach. The
HPLC method allows the GH activity of the toxin to be monitored in instances were the
fluorometric assay is not feasible. This assay is also adaptable to study other NADases or monoADP-ribosyltransferases that specifically possess an affinity tag like in PE24H presented here.
20
Acknowledgments
Supported by the Canadian Cystic Fibrosis Foundation (CCFF) and the Canadian
Institutes of Health Research (CIHR) grants to ARM, CCFF Doctoral Studentship to SPY. We
thank Dr. Norman J. Oppenheimer (Dept. of Pharmaceutical Chemistry, University of California,
San Francisco, CA) for the 2’-F-ribo-NAD+ gift.
References
1. S.R. Price, P.H. Pekala, Pyridine Nucleotide-Linked Glycohydrolases, in: D. Dolphin, O.
Avramovic, R. Poulson (Eds.) Pyridine Nucleotide Coenzymes: Chemical, Biochemical,
and Medical Aspects Part B, John Wiley & Sons, New York, 1987, pp. 513-548.
2. H. Kim, E.L. Jacobson, M.K. Jacobson, NAD glycohydrolases: a possible function in
calcium homeostasis, Mol. Cell Biochem. 138 (1994) 237-243.
3. P. Handler, J.R. Klein, The inactivation of pyridine nucleotides by animal tissues in vitro.,
J. Biol. Chem. 143 (1942) 49-57.
4. M. Ziegler, D. Jorcke, M. Schweiger, Identification of bovine liver mitochondrial NAD+
glycohydrolase as ADP-ribosyl cyclase, Biochem. J. 326 (1997) 401-405.
5. D.W. Chung, R.J. Collier, Enzymatically active peptide from the adenosine diphosphateribosylating toxin of Pseudomonas aeruginosa, Infect. Immun. 16 (1977) 832-841.
6. B.A. Wilson, K.A. Reich, B.R. Weinstein, R.J. Collier, Active-site mutations of diphtheria
toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine,
Biochemistry 29 (1990) 8643-8651.
7. S.L. Parikh, V.L. Schramm, Transition state structure for ADP-ribosylation of eukaryotic
elongation factor 2 catalyzed by diphtheria toxin, Biochemistry 43 (2004) 1204-1212.
8. C.M. Douglas, R.J. Collier, Exotoxin A of Pseudomonas aeruginosa: substitution of
glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity, J.
Bacteriol. 169 (1987) 4967-4971.
9. B.K. Beattie, G.A. Prentice, A.R. Merrill, Investigation into the catalytic role for the
tryptophan residues within domain III of Pseudomonas aeruginosa exotoxin A,
Biochemistry 35 (1996) 15134-15142.
10. B.K. Beattie, A.R. Merrill, A fluorescence investigation of the active site of Pseudomonas
aeruginosa exotoxin A, J. Biol. Chem. 274 (1999) 15646-15654.
21
11. S. Armstrong, A.R. Merrill, Application of a fluorometric assay for characterization of the
catalytic competency of a domain III fragment of Pseudomonas aeruginosa exotoxin A,
Anal. Biochem. 292 (2001) 26-33.
12. S.R. Blanke, K. Huang, R.J. Collier, Active-site mutations of diphtheria toxin: role of
tyrosine-65 in NAD binding and ADP-ribosylation, Biochemistry 33 (1994) 15494-15500.
13. P. Pietta, M. Pace, F. Menegus, High-performance liquid chromatography for assaying
NAD glycohydrolase from Neurospora crassa conidia, Anal. Biochem. 131 (1983) 533537.
14. S. Armstrong, J.H. Li, J. Zhang, A.R. Merrill, Characterization of competitive inhibitors for
the transferase activity of Pseudomonas aeruginosa exotoxin A, J. Enzyme Inhib. Med.
Chem. 17 (2002) 235-246.
15. B.A. Gruber, N.J. Leonard, Dynamic and static quenching of 1,N6-ethenoadenine
fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3(indol-3-yl) propyl) adenine, Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 3966-3969.
16. M. Li, F. Dyda, I. Benhar, I. Pastan, D.R. Davies, Crystal structure of the catalytic domain
of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog:
implications for the activation process and for ADP ribosylation, Proc. Natl. Acad. Sci. U.
S. A. 93 (1996) 6902-6906.
22
Figure Legends
Fig. 1. NAD+-glycohydrolase catalyzed reaction. (A) The reaction scheme. (B) Structure of
the NAD+ analogues F-NAD+ and -NAD+.
Fig. 2. Nicotinamide standard curve generated by HPLC. The peak area of nicotinamide
was calibrated by the peak area of the internal standard, (A) PABA or (B) N-acetyl-Lphenylalanine.
Fig. 3. Hydrolysis of NAD+ by NaOH measured by HPLC. (A) Chromatogram representing
the starting substrate (NAD+), reaction products (nicotinamide, ADP-ribose (ADPR)) and the
internal standard (PABA) at 5 min after the addition of base. (B) The reaction rate determined
from the time-course plot. The rate is 50 ( 1) pmoles of nicotinamide produced per minute.
Fig. 4. NAD+-glycohydrolase activity of PE24H determined using the fluorescence assay.
Wild-type PE24H (1.0 M) in buffer (open squares); buffer only (no toxin) (open circles); wildtype PE24H (1.0 M) in 75:25 v/v % mobile phase and buffer (open triangles); wild-type PE24H
(1.0 M) in 75:25 v/v % mobile phase (containing the internal standard PABA) and buffer
(stars). All samples contained 500 M -NAD+. See Materials and Methods for assay details.
Fig. 5. Hydrolysis of NAD+ by PE24H (NAD+-glycohydrolase activity). Chromatograms of
the hydrolysis of NAD+ at 0 (A) and 4 (B) hours. The toxin concentration was 10 M and NAD+
was 450 M. The reaction was monitored for 4 hours. All peaks are labelled and PABA is the
23
internal standard. (C) The reaction rate determined from the time-course plot. The rate is 64 (
7) M of nicotinamide is produced per hour.
Fig. 6. Inhibitory and binding properties of F-NAD+. (A) Dose-response curve for F-NAD+
on the ADPRT activity of PE24H. The assay included 20 mM Tris-HCl, pH 7.9, 500 M NAD+, 14 M eEF2, 0-400 M F-NAD+ and 5 nM PE24H in a total volume of 70 L. (B) The
binding of F-NAD+ (open squares) and NAD+ (open circles) to PE24H was determined using a
titration conducted in 20 mM Tris-HCl, pH 7.9, 50 mM NaCl at 25C with 1.25 M toxin. The
quenching of the intrinsic protein fluorescence was monitored with the fluorescence excitation
and emission at 295 and 340 nm, respectively (all band passes set to 4 nm). The data were
corrected for the dilution effect.
Fig. 7. Comparison of the rate of hydrolysis of F-NAD+ and NAD+ determined using an
HPLC-based NAD+-glycohydrolase assay. Chromatograms of F-NAD+ (425 M) hydrolysis
by PE24H (47.9 M) at 0 (A) and 48 (B) hours. Chromatograms of NAD+ hydrolysis are
represented in Fig. 5. All peaks are labelled and PABA is the internal standard. Rate of
hydrolysis of NAD+ (C) or F-NAD+ (D) determined by monitoring the ADPR product peak (see
text for explanation). Assay details are as described in Materials and Methods.
Fig. 8. Comparison of the rate of hydrolysis of -NAD+ determined using the HPLC-based
or fluorescence based NAD+-glycohydrolase assay. Chromatograms of -NAD+ (450 M)
hydrolysis by PE24H (10 M) at 0 (A) and 7 (B) hours. All peaks are labelled and N-Ac-L-Phe
24
is the internal standard. Rate of hydrolysis of -NAD+ determined by HPLC (C) or fluorescence
(D). Assay details are as described in Materials and Methods.
25
Fig. 1
nicotinamide
A.
O
O
+
H
+
N
O
-
O
O P
O
O P
OH
O
O
O
water
NH2
N
O
OH
H
N
O
-
O
O P
N
O
N
O P
OH
HO
OH
HO
O
N
O
OH
HO
B.
O
O
+
+
O
-
N
NH2
O
O
O P
O
O P
O
O P
F
HO
O
O
NH2
N
-
N
O
N
N
NH2
O
-
O
O P
OH
H
N
HO
O
O
N
-
+
N
O
N
N
OH
OH
HO
HO
F-NAD+
-NAD+
N
N
ADP-ribose
N
NH2
N
-
O
NAD+
O
NH2
O
HO
-
N
H
NH2
O
peak area of nicotinamide
calibrated with the internal standard
N-acetyl-L-phenylalanine
peak area of nicotinamide
calibrated with the internal standard
PABA
26
Fig. 2
0.8
A.
0.6
0.4
0.2
0.0
0
0
100
pmoles of nicotinamide
100
200
200
300
0.8
B.
0.6
0.4
0.2
0.0
300
pmoles of nicotinamide
400
500
400
500
27
Fig. 3
0.10
A.
+
NAD
Absorption Units
0.08
0.06
0.04
ADPR
0.02
PABA
nicotinamide
0.00
0
2
4
6
8
Retention Time (minutes)
pmoles of nicotinamide produced
250
B.
200
150
100
50
0
0
1
2
3
Time (minutes)
4
5
28
Fig. 4
250
Fluorescence Intensity (a.u.)
240
230
220
210
200
190
180
0
50
100
150
Time (minutes)
200
250
29
Fig. 5
300
A.
nicotinamide produced (M)
250
200
150
100
50
0
0
1
2
3
4
Time (hours)
0.08
0.07
B.
Absorption Units
0.06
NAD
+
0.05
0.04
0.03
0.02
PABA
0.01
0.00
0
2
4
6
8
Retention Time (minutes)
0.08
0.07
C.
Absorption Units
0.06
ADPR
0.05
0.04
0.03
NAD
+
0.02
0.01
PABA
nicotinamide
0.00
0
2
4
6
Retention Time (minutes)
8
30
Fig. 6
0.25
A.
Reaction Rate (M/sec)
0.20
0.15
0.10
0.05
0.00
0
100
200
300
400
+
[F-NAD ], M
Fractional Saturation (F/Fmax)
1.0
0.8
0.6
0.4
0.2
B.
0.0
0
200
400
+
600
+
[NAD or F-NAD ], M
800
1000
31
Fig. 7
0.06
0.06
A.
0.05
0.05
+
F-NAD
Absorption Units
Absorption Units
F-NAD
B.
0.04
0.03
0.02
0.01
0.03
0.02
0.01
ADPR
PABA
ADPR
nicotinamide
0.00
PABA
nicotinamide
0.00
0
2
4
6
8
0
2
Retention Time (minutes)
4
6
8
Retention Time (minutes)
0.40
3.0
C.
0.35
Calibrated ADPR Peak Area
2.5
Calibrated ADPR Peak Area
+
0.04
2.0
1.5
1.0
0.5
D.
D
0.30
0.25
.
0.20
0.15
0.10
0.05
0.0
0.00
0
1
2
Time (hours)
3
4
0
10
20
30
Time (hours)
40
50
32
Fig. 8
0.035
A.
-NAD
B.
0.030
+
Absorption Units
0.025
0.020
0.015
0.010
-NAD
+
0.025
0.020
0.015
0.010
N-Ac-L-Phe
0.005
-ADPR
0.005
nicotinamide
-ADPR
N-Ac-L-Phe
nicotinamide
0.000
0.000
0
2
4
6
8
10
12
14
0
2
4
Retention Time (minutes)
6
8
10
12
14
Retention Time (minutes)
80
70
C.
D.
350
60
D
300
M ADPR produced
M nicotinamide produced
Absorption Units
0.030
0.035
50
40
30
20
.
250
200
10
150
0
0
1
2
3
4
Time (hours)
5
6
7
0
1
2
Time (hours)
3
4