(Received for publication, January 23, 1995; and in revised form, March 28, 1995)
From the
The 3`:5`-cyclic nucleotide phosphodiesterase (CNP) of Vibrio fischeri, due to its unusual location in the periplasm,
allows this symbiotic bacterium to utilize extracellular 3`:5`-cyclic
nucleotides (e.g. cAMP) as sole sources of carbon and energy,
nitrogen, and phosphorus for growth. The enzyme was purified to
apparent homogeneity by a four-step procedure: chloroform shock,
ammonium sulfate precipitation, and chromotography on DEAE-Sephacel and
Cibacron Blue 3GA-agarose. The active enzyme consists of a single
polypeptide with a mass of 34 kDa. At 25 °C, it has a pH optimum of
8.25, a K
3`:5`-Cyclic nucleotide phosphodiesterase (EC 3.1.4.17;
CNP)
We recently described a second
extra-cytoplasmic CNP. The enzyme occurs in the marine bacterium Vibrio fischeri, which establishes a luminescent mutualism
with certain marine animals. Its periplasmic location and high activity
confer on V. fischeri the novel ability to utilize
extracellular 3`:5`-cyclic nucleotides as sources of carbon and energy,
nitrogen, and phosphorus for growth(6) . The gene, cpdP, for this enzyme is the only bacterial CNP gene that has
been cloned. The deduced amino acid sequence exhibits 34% identity with
the extracellular CNP of D. discoideum and 30% identity with
the low affinity CNP (PDE1) of the yeast Saccharomyces
cerevisiae(7) . The V. fischeri enzyme is
specific for 3`:5`-cyclic nucleotides (6) and, therefore,
differs from previously described bacterial periplasmic phosphatases
such as 2`:3`-cyclic phosphodiesterase:3`-nucleotidase (EC 3.1.4.16)
and 5`-nucleotidase (EC 3.1.3.5).
The V. fischeri enzyme
has been proposed to play a central role in the luminescent (light
organ) symbiosis of V. fischeri with sepiolid squids and
monocentrid fish(6) . Since periplasmic CNP activity is rare in
bacteria(6) , the enzyme may contribute to the specificity of
the symbiosis by permitting V. fischeri cells to utilize
putative host-released cAMP as a nutrient. Alternatively, the enzyme
may function to degrade 3`:5`-cyclic nucleotides released from
organisms into the marine environment(6, 7) .
In
this report, we describe the purification and biochemical properties of
the V. fischeri periplasmic CNP. A detailed understanding of
the enzyme may lead to insights into the physiological and ecological
roles of bacterial CNPs and the possibility of cAMP-mediated symbiotic
interactions between V. fischeri and its hosts.
Control experiments demonstrated that the reaction
rates obtained with assay 1 were linearly dependent on the amount of
CNP in the reaction mixture over a 4-fold range (0.04-0.164
µmol of NADH oxidized/min), indicating that the observed CNP
activity was not limited by any of the rate indicator components. This
finding was confirmed at each temperature used in the determinations of K
Assay 2,
a two-stage version of assay 1, was developed to examine the effects of
pH, temperature, and metal ions on CNP activity without interference of
these effects on the activities of the coupling enzymes. In the first
stage, samples containing CNP were added to reaction mixtures
containing 100 mM Tris-HCl, pH 8.25, and 6 mM cAMP in
a volume of 1.0 ml; the reactions were initiated by the addition of the
CNP and were terminated at various times by the addition of 53 µl
of 60% (w/v) perchloric acid, which irreversibly inactivated the CNP.
This mixture was placed on ice, and 142 µl of 3 M KOH was
then added to raise the pH to approximately 6 and to precipitate the
perchlorate anion. In the second stage, 20 µl of the
perchlorate/KOH-treated reaction mixture was added to a mixture
containing the components of Assay 1 except for the CNP and cAMP. The
total
Assay 3,
used for substrates other than cAMP, was a modification of a previously
described method (6, 12) with the following additional
changes: 100 mM Tris-HCl, pH 8.25 at 25 °C as the reaction
buffer, without MnCl
The periplasmic extract
was fractionated with ammonium sulfate, and the protein that
precipitated between 40 and 70% of saturation was collected by
centrifugation (16,000
The dialyzed ammonium sulfate fraction was applied to 2 ml
of packed DEAE-Sephacel equilibrated with buffer 2 in a 10-ml Poly-Prep
column (Bio-Rad). 10 ml of 50 mM NaCl in buffer 2 was run
through the column and discarded. CNP was then eluted from the column
with 10 ml of 100 mM NaCl in buffer 2. The entire eluate was
immediately applied to 1 ml of Cibacron Blue-agarose equilibrated with
100 mM MgSO
Figure 1:
SDS-PAGE analysis during purification
of the V. fischeri periplasmic CNP. PE, periplasmic
extract; AS, 40-70% ammonium sulfate fraction; DS, DEAE-Sephacel eluent; BA, Cibacron Blue-agarose
eluent; and, PS, protein standards (66 kDa, bovine serum
albumin; 45 kDa, ovalbumin; 36 kDa, glyceraldehyde-3-phosphate
dehydrogenase; 29 kDa, carbonic anhydrase; 24 kDa, trypsinogen; 20.1
kDa, trypsin inhibitor; 14.2 kDa,
In the third step, the resolubilized ammonium sulfate
fraction was bound to and eluted from DEAE-Sephacel, resulting in a
25-fold purification. Earlier purification attempts using buffer 2 in
this anion-exchange step at a pH of 6.0, which is close to the
protein's calculated pI of 5.5(7) , were successful but
substantial losses of activity occurred. Therefore, the enzyme is
apparently unstable at low pH. Adjusting buffer 2 to pH 7.5 (Table 1) eliminated the loss of activity.
In the final step,
in which the DEAE-Sephacel eluate was bound to and eluted from Cibacron
Blue-agarose, a 26-fold purification was achieved. The simplicity of
the purification procedure overall results from the high affinity of
the protein for the blue-agarose dye matrix in this step. The high
affinity permits the extreme conditions of high KCl concentration and
high pH to be used to elute the majority of contaminating proteins.
Adenosine, a competitive inhibitor of the V. fischeri CNP with
a K
Figure 2:
Native molecular weight determination of V. fischeri periplasmic CNP. One of three experiments is shown
in which proteins were separated on Sephacryl HR200. CNP (32.4 kDa, Log
molecular weight = 4.51) purified as described in the text
(
Figure 3:
Effect of pH on hydrolysis of cAMP by V. fischeri periplasmic CNP. Assay 2 was used, and the buffers
were 50 mM glycine with 50 mM glycyl-glycine (
Figure 4:
Effect of temperature on hydrolysis of
cAMP by V. fischeri periplasmic CNP. The assay mixture was
adjusted to pH 8.25 at each temperature. Data presented are the
averages of duplicate assays. A, Hofstee (16) plots
were used to calculate values of K and V
Reactivation of zinc-free apoenzyme with various metal
ions supported this conclusion. The apoenzyme was prepared by dialysis
of the purified protein against EDTA, followed by size exclusion
chromatography to separate EDTA
Figure 5:
Inactivation of the V. fischeri periplasmic CNP by EDTA and reactivation by metal ions. A, activity of CNP dialyzed against buffer containing 1 mM EDTA (
Purification of the the periplasmic CNP of V. fischeri to apparent homogeneity permitted the characterization of some of
its properties. Several of these, including its narrow substrate
specificity, stability, abundance in the cell, specific activity, and
the ability of certain metal ions to reactivate apoenzyme are unusual
and provide insight into the role of the enzyme in the biology of V. fischeri and the ecology of cyclic nucleotides in seawater.
The narrow substrate specificity of the V. fischeri CNP is
atypical for a periplasmic nucleotidase. Other nucleotide degrading
periplasmic phosphatases, such as alkaline phosphatase, acid
phosphatases, 2`:3`-cyclic phosphodiesterase:3`-nucleotidase, and
UDP-glucose hydrolase:5`-nucleotidase, are each capable of hydrolyzing
multiple types of phosphorylated compounds, but the CNP of V.
fischeri is specific for 3`:5`-cyclic nucleotides; it shows no
activity with 2`:3`-cAMP, ADP, ATP, or non-nucleotide phosphate esters (6) . Activity measurements with various 3`:5`-cyclic
nucleotides suggest that the 2`-oxygen of the ribose moiety is
necessary for full activity of the enzyme, and that the enzyme does not
discriminate between purines and pyrimidines (Table 2).
Despite its narrow substrate specificity, the V. fischeri enzyme is similar to many periplasmic phosphatases in containing
zinc. Alkaline phosphatase(18) , 2`:3`-cyclic
phosphodiesterase:3`-nucleotidase(19) , and UDP-glucose
hydrolase:5`-nucleotidase (19) are zinc metalloenzymes.
However, there appears to be no significant amino acid sequence
similarity between the four proteins. Other than the low affinity CNP
(PDE1) of S. cerevisiae, which also contains 2 atoms of
zinc/peptide(20) , no eukaryotic CNPs have been shown to be
metalloenzymes.
The mature CNP protein has no cysteine
residues(7) , and, therefore, no disulfide bonds to stabilize
its tertiary structure. Nonetheless, several observations in this
report indicate that it is a very stable protein. Full catalytic
activity was retained after exposure of the enzyme to 60 °C (Fig. 4B) or to a pH of 10.3 (as used in the
dye-affinity step of the purification procedure). In addition, the
binding of one or both of the zinc ions is tight, since prolonged
dialysis against EDTA was required to inactivate the CNP; after 24 h of
dialysis, the activity was 75% of the starting value, and a period of
140 h was required to reduce the activity by 99% (Fig. 5A). A prolonged dialysis against EDTA is also
required to inactivate the zinc-containing CNP of S.
cerevisiae(20) , and it is possible that the zinc ions in
both the yeast CNP and that from V. fischeri are firmly bound
to the same ligands (see below).
Copper, magnesium, and, calcium,
are all unusual substitutes for zinc, but in metal substitution
experiments with the V. fischeri enzyme, activity was
partially restored to the apoenzyme by each of these metal ions. Copper
has been used as a spectroscopic probe in metal substitution
experiments, but with the notable exception of superoxide dismutase,
copper-substituted zinc enzymes usually are inactive(21) .
Similarly, glyoxylase is one of a few zinc enzymes that is active when
substituted with magnesium(22) . Yeast enolase has been shown
by x-ray diffraction to bind zinc or calcium at the same site as the
native magnesium ion, but the calcium-substituted enzyme is inactive
due to an alteration in the conformation of bound substrate (23) . The difficulty in removing zinc from the V. fischeri CNP and the reactivation of the apoenzyme by calcium, which is
surprising because calcium has a valence electronic configuration
distinct from that of zinc, may indicate that zinc serves as a
structural, rather than a catalytic, component in CNP.
However,
contrary to the proposal that the zinc of CNP is structural, a recent
analysis of zinc enzymes with known structures (24) suggests
that the zinc might be catalytic. In that study, it was pointed out
that non-catalytic zinc has 2 or more cysteine residues as ligands, of
which the mature V. fischeri CNP has none. In addition,
catalytic zinc often has 1 or more histidine residues as ligands, of
which the CNP has 4 that, by sequence comparison, are evolutionarily
conserved(7) . Determination of the role of zinc in the CNP of V. fischeri will require further examination.
The rapid
kinetics of the V. fischeri enzyme may make it an interesting
enzyme for studies of catalytic mechanism. Using the turnover number
and K
The pH/activity profile (Fig. 3)
supports the involvement of 1 or more histidine residues in CNP
activity. Although side chain ionizable groups on amino acid residues
in proteins often have pK
The biochemical characteristics of
the V. fischeri CNP are consistent with hypotheses for the
biological role of the enzyme in the ecology and symbiosis of V.
fischeri. The enzyme has been proposed to degrade cAMP free in the
environment(6) . Many organisms release
cAMP(30, 31) , but the concentration in the
environment is low (31, 32) . The CNP of V.
fischeri, because of its periplasmic location and high specific
activity, could function in a manner analogous to phosphate scavenging
periplasmic enzymes in bacteria. Many bacteria have periplasmic
5`-nucleotidase activity, which degrades AMP to inorganic phosphate and
adenosine prior to their transport into the cytoplasm and subsequent
metabolism. The V. fischeri CNP, therefore, represents a novel
class of enzyme that can catalyze the first step in the recovery of
cAMP from the environment.
In the luminescent mutualism of V.
fischeri with sepiolid squid and monocentrid
fish(33, 34) , the enzyme has been hypothesized to
permit V. fischeri, exclusively, to utilize host-provided cAMP
as a source of nutrition. According to the hypothesis, V. fischeri cells in the animal light organ elicit the overproduction and
release of host cAMP by secreting a toxin that, through an
ADP-ribosylating activity, interferes with the regulation of host
adenylate cyclase in a manner analogous to the secretion and activity
of cholera enterotoxin by V. cholerae in the human intestine (6, 7, 35) . Recent evidence indicating that V. fischeri contains toxRS genes (36) is
consistent with this hypothesis. Nonetheless, a direct test of the
symbiosis hypothesis will require examination of the ability of a cpdP null mutant of V. fischeri to colonize its
animal host.
We thank R. Auxier of the Chemical Analysis
Laboratory, University of Georgia, for conducting the ICPES metal
content analysis, L. Ball of the Inductively Coupled Plasma Facility at
the Woods Hole Oceanographic Institution for technical assistance and
for providing the Specpure Analytical Standard salts, and L. Gilson for
comments on the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
for cAMP of 73 µm, and a V
of 3700 µmol of cAMP hydrolyzed/min/mg
protein (turnover number of 1.24
10
/min). The
specific activity of the V. fischeri enzyme is approximately
20-fold greater than that of any previously characterized CNP when
comparisons of activity are made at the same assay temperature.
Activity increases with temperature up to 60 °C. The CNP contains 2
atoms of zinc/monomer, and zinc, copper, magnesium, and calcium can
restore activity of the apoenzyme to varying degrees. The exceptional
specific activity of the enzyme and its unusual location in the
periplasm support proposals that the enzyme enables the bacterium to
scavenge 3`:5`-cyclic nucleotides in seawater and that the enzyme plays
a role in cAMP-mediated host-symbiont interactions.
(
)catalyzes the hydrolysis of 3`:5`-cyclic
nucleotides (e.g. cAMP and cGMP) to their corresponding
5`-nucleoside monophosphates. The enzyme typically is located in the
cytoplasm of cells. In eukaryotic organisms, several isozymes function
in a variety of signal-mediated processes by modulating cytoplasmic
levels of cAMP and cGMP(1) . Many prokaryotes also produce a
cytoplasmic CNP. In bacteria, cAMP regulates gene transcription via
interaction with a cAMP receptor protein. However, rather than
CNP-mediated hydrolysis of cAMP or excretion from the cytoplasm,
regulation of the synthesis of cAMP is thought to control cellular
levels in bacteria(2, 3) . A CNP with atypical
locations in the cell has been described from the cellular slime mold Dictyostelium discoideum. That enzyme is unique in occurring
as both extracellular and cell membrane-associated forms, which
catalyze the hydrolysis of extracellular cAMP involved in morphogenetic
and aggregational signaling during plasmodium
formation(4, 5) .
Reagents
Myokinase (360 units/mg), pyruvate
kinase (200 units/mg), lactate dehydrogenase (550 units/mg), and
crystalline bovine serum albumin were purchased from Boehringer
Mannheim. 5`-Nucleotidase (Crotalus adamanteus venom), 5`-ATP
(disodium salt), NADH (disodium salt), 3`:5`-cyclic nucleotides, Trizma
grade tris, phosphenolpyruvate (tri-cyclohexylammonium salt), Cibacron
Blue-agarose 3GA, DEAE-Sephacel, and Sephacryl HR200 were obtained from
Sigma, and Coomassie R-250 was from Fisher Chemical Co. (Pittsburgh,
PA).
Bacterial Strains and Culture Conditions
V.
fischeri strain MJ-1, from the light organ of the monocentrid fish Monocentris japonicus(8) , was maintained on LBS agar (6) at room temperature. Liquid cultures were grown at 26
°C with aeration (150 revolutions/min) in minimal medium containing
300 mM NaCl, 10 mM KCl, 50 mM MgSO, 10 mM CaCl
, 15 mM NH
Cl, 0.3 mM
-glycerophosphate, 20
mg/liter ferric ammonium citrate, 10 mM glucose, and 100
mM HEPES, pH 7.5. Cultures for enzyme purification were
initiated with a 1% inoculum that had been grown to mid-exponential
phase in minimal medium. Cells were harvested by centrifugation
approximately 3 h after cultures attained stationary phase, at which
point they produced a high level of luminescence.
Cell Fractionation
Cells were separated into outer
membrane, inner membrane, and cytoplasmic/periplasmic (i.e. soluble) fractions as described for Vibrio
cholerae(9) .
CNP Activity Assays
Three assays were used for
measurement of CNP activity. In assay 1, the standard method used in
this study, rates of hydrolysis of cAMP to AMP were measured by a
modification of a coupled assay with adenylate kinase, pyruvate kinase,
and lactate dehydrogenase(10) . Specifically, identical
reference and reaction mixtures consisted of 2 mM cAMP (except
where otherwise indicated), 0.6 mM ATP, 0.17 mM NADH,
0.5 mM phosphoenolpyruvate, 4 units of adenylate kinase, 4
units of pyruvate kinase, 10 units of lactate dehydrogenase, 20
mM KCl, 5 mM MgCl, and 100 mM Tris-HCl, pH 8.25, in a volume of 2 ml. The reaction was initiated
by addition of samples containing CNP, and the production of
NAD
from NADH was monitored spectrophotometrically as
the change in absorbance at 340 nm. Consistent with the coupled nature
of the assay, the reaction exhibits a lag that is inversely
proportional to the amount of CNP added. Following the lag, the
reaction rate is linear up to a
A
of 0.8.
CNP activity was calculated from the linear portion of the reaction
using the extinction coefficient of 6.22/mM/cm (11) taking into account that 2 mol of NAD
are
produced per mole of cAMP hydrolyzed. One unit of CNP activity is
defined as the activity that catalyzes the hydrolysis of 1 µmol of
cAMP in 1 min.
and V
.
Furthermore, since adenosine was found to be a competitive inhibitor of
CNP (see ``Results''), control experiments were conducted and
verified that ATP (included in the assay at 0.6 mM as an
adenylate kinase substrate) does not inhibit CNP activity.
A
was used to calculate the amount of cAMP
hydrolyzed to AMP during the first-stage reaction. Rates were linear
with respect to the time of the first reaction. Rate measurements made
with assay 2 agreed within 10% of those made with assay 1.
which was not required for V.
fischeri CNP activity, and with a 5`-nucleotidase reaction time of
30 min. Unless otherwise indicated, assays 1-3 were conducted at
25 °C. Protein concentrations were determined by the method of
Bradford (13) using bovine serum albumin as the standard.
Purification
Periplasmic proteins were released
from intact V. fischeri cells by a chloroform shock
method(14) . Cells from two 1-liter cultures were collected by
centrifugation (10,000 g, 10 min) and resuspended in
100 ml of 50 mM Tris-HCl, pH 8.0 (buffer 1) at 4 °C. 20 ml
of chloroform was added to the cell suspension, which was inverted once
to mix and then held at room temperature for 10 min. An additional 200
ml of buffer 1 was added, after which cells and chloroform were
pelleted by centrifugation (16,000
g, 20 min, 4
°C). Buffer 1 was added to the supernatant (periplasmic extract) to
adjust its protein concentration to 1 mg/ml. All subsequent steps of
the purification were conducted at 4 °C.
g, 10 min) and dissolved in 10
ml of 10 mM imidazole buffer, pH 7.5 (buffer 2). The protein
solution was then dialyzed for 18 h against three 1-liter volumes of
buffer 2, changed at 0, 6, and 12 h, to remove the residual ammonium
sulfate.
in a second 10-ml Poly-Prep column. An
initial wash with 10 ml of 1 M KCl, 50 mM Tris base
(untitrated, pH 10.3; buffer 3) removed the majority of bound protein,
and CNP was then eluted with 5 ml of buffer 3 containing 1 mM adenosine. At this point, aliquots were taken for SDS-PAGE
analysis, activity assays, and protein determination. To preserve
activity, crystalline bovine serum albumin was added to 5 mg/ml to the
remaining sample. The adenosine was removed by 18 h of dialysis with
three 1-liter changes of 1 M KCl and 50 mM Tris-HCl,
pH 9.0. Without the addition of bovine serum albumin, CNP activity
declined during storage at -80 °C. With bovine serum albumin,
no loss of activity was detected after 12 months of storage at
-80 °C with repeated thawing and freezing.
Molecular Weight Determinations
For SDS-PAGE, the
method of Laemmli (15) was followed using 14% separating and 6%
stacking gels. Proteins were stained with Coomassie Blue R-250 at 60
°C. For gel-filtration chromatography, a 1.5 50-cm column
containing Sephacryl HR200 was used with 1 M KCl buffered with
100 mM Tris-HCl, pH 9.0, at 4 °C at a flow rate of 19
ml/h. Fractions were collected each minute and assayed for CNP activity
by assay 1.
Inactivation of CNP by EDTA
A 300-µl aliquot
of purified CNP solution containing 5 mg/ml bovine serum albumin was
dialyzed for 140 h against three 100-ml volumes of 1 M KCl, 1
mM EDTA, and 50 mM Tris-HCl, pH 9.0, at 4 °C
changed at 0, 48, and 96 h. After dialysis, 1% of the starting enzyme
activity remained. The EDTA was then removed by size exclusion
chromatography using a Bio-Spin 6 Column (Bio-Rad) prewashed with 1
mM EDTA and re-equilibrated with deionized water according the
manufacturer's instructions. Nine divalent metal ions (Specpure
Analytical Standard salts (Johnson Matthey, Ward Hill, MA)) were added
to reaction mixtures for the first stage of assay 2 at concentrations
of 1, 10, and 100 µM to test for their ability to
reactivate the apoenzyme. Those metals found to reactivate the
apoenzyme were examined further to determine their optimal
concentrations for reactivation. Percent reactivation of the apoenzyme
was calculated as: [(activity of apoenzyme with metal ion
addition) minus (activity without metal ion
addition)]/[activity of control without EDTA]
100.
Metal Content Determination
ICPES analysis of the
purified CNP was performed by the Chemical Analysis Laboratory,
University of Georgia (Athens, GA) using the Thermo Jarrell Ash 965
Inductively Coupled Argon Plasma with simultaneous 31 element
capability. The CNP was tested for the presence of 31 elements
including calcium, cadmium, cobalt, copper, iron, magnesium, manganese,
nickel, and zinc which are most frequently found as components of
metalloproteins. The metal content of CNP samples was corrected for
metal content in control samples containing buffer alone.
Periplasmic Location
Previously, we demonstrated
the CNP activity of V. fischeri to be soluble in the
periplasm(6) . That study, however, did not address the
possibility that the enzyme also occurs in a cytoplasmic membrane- or
outer membrane-associated form or that it is released into the
extracellular environment. To test these possibilities, we examined
membrane and soluble fractions of V. fischeri cells and the
cell-free growth medium for CNP activity. Cells were separated into
outer-membrane, cytoplasmic membrane, and cytoplasm/periplasm (soluble)
fractions. Total cellular CNP activity before fractionation was 291
units, and the fractions were found to contain 0.5, 6, and 224 units,
respectively, demonstrating that the enzyme is not associated with
either the outer membrane or the cytoplasmic membrane. No CNP activity
was observed in the cell-free growth medium following growth of V.
fischeri cells to high population density or in protein
precipitated from the cell-free growth medium by the addition of
ammonium sulfate to 80% of saturation. The enzyme, therefore, does not
appear to be released into the extracellular environment.
Purification
A four-step procedure was developed
that resulted in purification of the enzyme by approximately 4000-fold (Table 1) and to apparent homogeneity (Fig. 1). The first
step, chloroform shock, served to disrupt the outer membrane and
release the periplasmic contents from the cells, giving an
approximately 3-fold increase in specific activity relative to whole
cells. No cell lysis was detected by phase contrast microscopy when
cells were mixed gently with the chloroform. Vigorous mixing of cells
with the chloroform eliminated most of the CNP activity, as did
freezing of the sample at any step in the purification procedure before
it was complete.
-lactalbumin).
The second step, fractionation of the periplasmic
suspension with ammonium sulfate, gave a 2-fold increase in specific
activity. It also served to concentrate the sample for the two
subsequent chromatographic steps, in which most of the purification was
achieved.
of approximately 800 µM (data not shown), was used at a concentration of 1 mM in
the wash buffer to elute CNP from the blue-agarose. SDS-PAGE analysis
of the adenosine eluate revealed a single protein, even when the gel
was heavily loaded (Fig. 1). The enzyme was estimated to account
for approximately 0.025% of the total cellular protein and 0.07% of the
periplasmic protein. Previously, the enzyme purified in this manner was
subjected to microsequence analysis of the first 20 residues at the
amino-terminal end, and the sequence for the mature protein matched
that deduced from the cloned gene(7) .
Molecular Mass of the Native CNP
The purified
protein exhibited a monomeric molecular mass of approximately 34,000 Da
on SDS-PAGE (Fig. 1), which is consistent with the mass of the
protein calculated from the deduced amino acid sequence of the cloned cpdP gene (33,636 for the protein without its leader sequence;
7). To determine if the enzyme was active as a monomer, we used a
calibrated Sephacryl gel filtration column to conduct size exclusion
chromatography of the purified protein under non-denaturing conditions.
The CNP activity in each of three trials eluted as a single molecular
species, with an estimated molecular mass of 32,000 ± 800 Da
(mean ± S.D.). A representative experiment is shown in Fig. 2. The similarity of the estimated masses under denaturing
and non-denaturing conditions indicates that the native enzyme is
active as a monomer and may be present in the periplasm as such.
) and protein size standards (
) (160 kDa, aldolase; 106
kDa, 6-phosphogluconate dehydrogenase; 66 kDa, bovine serum albumin; 45
kDa, ovalbumin; 29 kDa, carbonic anydrase; 21 kDa, adenylate
kinase).
cAMP Hydrolysis: Kinetics, pH, and Temperature
Effects
Activity of the purified periplasmic protein was maximal
at pH 8.25, with half-maximal activities at pH values of approximately
7.0 and 9.0 (Fig. 3). The enzyme obeys typical Michaelis-Menten
kinetics and has a Kfor cAMP of 73
µM at pH 8.25 and 25 °C (Fig. 4A), as
derived from a Hofstee plot(16) . V
at
25 °C, calculated from the Hofstee plot (Fig. 4A),
was 3700 units/mg protein, and the measured rate at 2 mM cAMP
was approximately 3400 units/mg. The corresponding turnover number
using the calculated molecular mass of 33,636 is 1.24
10
/min.
)
and 100 mM MES (
). Data presented are the averages of
duplicate assays. For each data point, the measured values differed by
<5% from the average.
for cAMP hydrolysis at various temperatures: 5 °C, K = 53 µM and V
=
588 units/mg (
); 15 °C, K = 59 µM and V
= 2077 units/mg (
); 25
°C, K = 73 µM and V
= 3730 units/mg (
); 35 °C, K =
132 µM and V
= 8764 units/mg
(
). For each data point, the measured values differed by <3%
from the average. B, plot of measured specific activities at
different temperatures. For each data point, the measured values
differed by <5% from the average.
It was of interest to determine if the CNP
exhibited maximum activity at a temperature that the bacterium
experiences in seawater. Enzymatic reactions in V. fischeri,
as in many marine plants and poikilothermic animals, generally occur at
temperatures of approximately 27 °C or lower. Remarkably, the rate
of the CNP-catalyzed reaction increases with temperature up to 60
°C (Fig. 4B) at which temperature the measured
specific activity was about 27,000 units/mg, indicating that the CNP is
active at temperatures much higher than it is expected to experience in
nature. K and V
were determined at 10 °C intervals between 5 and 35 °C (Fig. 4A). The K
for cAMP
increased from 53 µM at 5 °C to 132 µM at
35 °C, and V
increased by 15-fold over the
same range, giving a coefficient of 2.47/10 °C rise in temperature.
Substrate Specificity
Previously, we demonstrated
that the V. fischeri periplasmic CNP is specific for
3`:5`-cyclic nucleotides(6) . Consistent with that study, cGMP
was utilized at approximately half the rate of cAMP (Table 2).
The rates observed with cCMP, cIMP, and cUMP were similar to or
somewhat higher than that with cAMP, whereas those with the
2`-deoxy-cyclic nucleotides, 2`-deoxy-cAMP and cTMP, were 3-fold lower.
Metal Content
Our earlier observation that EDTA
inhibits activity (6) suggested that the CNP might be a
metalloenzyme or might require a divalent metal ion cofactor for full
activity. Furthermore, sequence similarity of the V. fischeri enzyme to a known zinc enzyme, the low affinity CNP (PDE1) of S. cerevisiae, including the conservation of 4 histidine
residues in the two sequences (7) , suggested that the V.
fischeri enzyme contains zinc. Therefore, we tested three
chelators of zinc for their effects on CNP activity: EDTA,
dithiothreitol, and 1,10-orthophenanthroline. The chelators, present in
the reaction mixture at 1 mM, inhibited CNP activity 23, 64,
and 98%, respectively. With regard to inhibition by dithiothreitol, a
strong chelator of zinc(17) , it should be noted that the
mature CNP protein has no cysteine residues(7) ; thus, the
inhibition by dithiothreitol cannot be due to effects on sulfhydryl
groups or disulfide bonds. Subsequent analysis by ICPES detected 2.2
mol zinc/mol CNP, while none of the other 30 elements tested was
detected in significant quantities. We conclude that the native V.
fischeri CNP is a zinc-containing enzyme, with two atoms of
zinc/peptide.
metal complexes from the
apoenzyme. 140 h of dialysis was required to reduce CNP activity to 1%
that of a control sample dialyzed against buffer alone, which retained
94% of its original activity (Fig. 5A). After removal
of EDTA from the sample, nine divalent metal ions,
Ca
, Cd
, Co
,
Cu
, Fe
, Mg
,
Mn
, Ni
, and Zn
,
were added to the apoenzyme to test their ability to restore activity.
Four metal ions reactivated the apoenzyme to varying degrees (Fig. 5B). Zinc was the most effective; 1 µM restored 94% of the activity of the control sample. Concentrations
above 1 µM were less effective or inhibitory. Copper (1
µM), magnesium (10 mM), and calcium (50
µM) restored 33, 47, and 67% of the activity,
respectively. Magnesium was still increasing enzyme activity at 10
mM, the highest concentration tested.
) and against buffer without EDTA (
). B,
activity of EDTA-inactivated CNP upon addition of various metal ions
(Zn
(
), Ca
(
),
Cu
(
), Mg
(
)). Data
presented are the average of duplicate assays. For each data point, the
measured values differed by <8% from the
average.
To test for the
involvement of a loosely bound metal ion in CNP activity, metal ions
were added to the reaction mixture with holoenzyme that had not been
exposed to EDTA. Of the nine metal ions tested with the apoenzyme, none
enhanced activity of the holoenzyme at a concentration of 100
µM, whereas Zn and Cu
inhibited holoenzyme activity about 25% and Cd
inhibited 77%. No inhibition was observed with calcium or
magnesium.
determined at 25 °C (Fig. 4A), k
/K
can be
calculated to be 2.8
10
s
M
, which indicates that the rate of
formation of the enzyme-substrate complex is nearly as fast as allowed
by diffusion controlled encounters between CNP and cAMP(25) .
At 30 °C, the assay temperature used in studies of other CNPs, the V. fischeri enzyme has a specific activity of 7100 units (Fig. 4B), a value more than 20 times greater than any
previously reported for a CNP that is specific for 3`:5`-cyclic
nucleotides, including that of the extracellular CNP of D.
discoideum(26) and the low affinity CNP of S.
cerevisiae(20) to which it has substantial amino acid
sequence identity(7) . The highest previously reported specific
activity for a CNP is that from bovine brain and heart, 300
units/mg(27) . The highest specific activity for catalysis of
cAMP hydrolysis by a CNP from a bacterium other than V. fischeri is that from Serratia marcescens, 295
units/mg(28) .
values that are
different from those seen with free amino acids, and the ionizable
groups involved in enzyme catalysis cannot be definitively identified
from a pH/activity profile, such profiles can be suggestive, especially
if additional information about the enzyme's structure and
function is available. The V. fischeri CNP exhibits
half-maximal activity at pH values of 7 and 9. Imidazole side chains of
histidine residues have pK
values in the
range of 5-8(29) , and 4 histidines, which occur
infrequently in bacterial proteins, are conserved in the CNPs of V.
fischeri, S. cerevisiae, and D. discoideum(7) .
Such highly conserved residues often have essential roles in enzyme
structure and/or catalysis. Thus, we assume that further study may show
that 1 or more of the histidines in V. fischeri CNP must be in
the unprotonated state for most efficient catalysis. Likewise with
regard to the half-maximal activity at pH 7, zinc-bound water is a
participant in many hydrolytic reactions, and it ionizes with a
pK
of about 7. Our data are also
consistent with a role for zinc-bound water in the activity of CNP. The
implications of the half-maximal activity at pH 9 are less clear.
Protein
-amino groups, lysine
-amino groups, and tyrosine
phenolic hydroxyl groups generally ionize with pK
values of about 9, and our results (Fig. 3) might
indicate involvement of one or more of these groups in the
CNP-catalyzed hydrolysis of cAMP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.