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(Received for publication, October 16, 1995; and in revised form, December 8, 1995) From the
2`,5`-Dideoxyadenosine 3`-di- and triphosphates were tested as
inhibitors of brain adenylyl cyclases. With an IC
Adenylyl cyclase is a family of membrane-bound enzymes that
catalyze the formation of 3`:5`-cAMP from 5`-ATP. In mammalian systems,
the 10 known adenylyl cyclase isozymes are regulated by numerous
hormones and neurotransmitters via cell surface receptors linked via
stimulatory (G Although the three-dimensional structure of adenylyl cyclase is not
known, the deduced primary sequence suggests a membrane topology
exhibiting a repeated structure of six membrane spanning regions
followed by a large cytosolic domain, giving twelve membrane spanning
regions and two cytosolic domains(19) . The two cytosolic
domains (C1 and C2) are homologous with each other and with the
established catalytic domain of guanylyl cyclases(20) ,
supporting the idea that each contains a nucleotide binding region.
However, it is not known whether C1, C2, or both form the catalytic
site, nor whether one or both domains participate in inhibition by
P-site ligands. Inhibition kinetics and irreversible inactivation
studies with P-site-selective covalent affinity probes are consistent
with inhibition occurring at a site that is distinct from the catalytic
site (13, 14, 15) . ( The most potent inhibitory ligands have been
2`,5`-dideoxyadenosine 3`-monophosphate (2`,5`-dd-3`-AMP) (
2`,5`-Dideoxyadenosine 3`-phosphates are a family of
inhibitors of adenylyl cyclase in which potency increased with the
number of 3`-phosphates (Fig. 1). IC
Figure 1:
Inhibition of rat brain adenylyl
cyclase by 2`,5`-dideoxyadenosine and its 3`-phosphorylated
derivatives. Activities were determined in the presence of 5 mM MnCl
2`,5`-dd-3`-ATP (IC Structurally, 2`,5`-dd-3`-ATP and 2`,5`-dd-3`-ADP share some
properties with 5`-ATP. To ascertain whether either 3`-nucleotide might
inhibit activity through simple competition with 5`-ATP at the
catalytic site, inhibition kinetics were evaluated (Fig. 2). In
this experiment, inhibition by 2`,5`-dd-3`-ATP of the forskolin
affinity-purified enzyme from bovine brain was found to be
noncompetitive with respect to substrate. Moreover, inhibiton was
linear (Fig. 2, inset) in that replots of slopes and
intercepts were also linear with inhibitor concentration. This linear
noncompetitive behavior was also seen with the crude,
detergent-extracted adenylyl cyclase from rat brain (not shown) and
also with 2`,5`-dd-3`-ADP (not shown). This behavior argues strongly
that inhibition occurred at a site distinct from catalysis and is fully
consistent with inhibition occurring at the P-site. P-site-mediated
inhibition of adenylyl cyclases is characteristically noncompetitive
with respect to substrate, whether Mn
Figure 2:
Kinetics of inhibition of purified bovine
brain adenylyl cyclase by 2`,5`-dd-3`-ATP. Activities were determined
in the presence of 5 mM MnCl
The straightforward and linear kinetic behavior
was somewhat surprising with the cruder detergent extracts as we had
expected that phosphohydrolases might have contributed to significant
breakdown of the 3`-polyphosphates(21) . However, we noted no
meaningful breakdown of either nucleotide during assay incubations
either with purified or with the crude Lubrol PX-extracted adenylyl
cyclase. The inhibitory potency of 2`,5`-dd-3`-ATP suggests that
derivatives of it may be useful as a labeled ligand to probe the P-site
binding domain on adenylyl cyclase. This could circumvent one of the
major constraints in the identification of amino acids involved in the
binding of P-site ligands. Presently one must rely on inhibition of
catalysis, even for evaluating efficacy of site-directed mutations of
expressed protein. With inhibition as end point one is by definition
not directly quantifying a single domain on this enzyme, but is rather
measuring a downstream event. Changes in catalysis could occur
independent of changes in the binding of inhibitory ligand, simply
through modification of a domain that influences coupling between
inhibitory and catalytic domains. The affinity of 2`,5`-dd-3`-ATP is
sufficient to allow a binding assay to be developed, and this could
significantly aid studies in this direction.
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2380-2382
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
40
nM, 2`,5`-dideoxy-3`-ATP is the most potent non-protein
synthetic regulator of adenylyl cyclases thus far described. Neither
2`,5`-dideoxy-3`-ADP nor 2`,5`-dideoxy-3`-ATP inhibited activity by
competition with substrate, and the linear noncompetitive inhibition
observed was consistent with interaction via a distinct domain. The
availability of this ligand will permit the development of a variety of
probes that will be extremely useful in investigating adenylyl cyclase
structure and the role(s) that this class of compound may play in
physiologically regulating cell function.
) and inhibitory (G
) guanine
nucleotide-dependent regulatory proteins (G-proteins), as well as by
numerous other agents also of physiological and biochemical interest.
These include agents or enzymes that act on hormone receptors, specific
bacterial toxins that act on G and G, and
agents that act directly on adenylyl cyclase. Direct activation can be
caused by forskolin with all but one isozyme and by
Ca/calmodulin with four isozymes. The enzyme is also
inhibited directly by certain adenosine derivatives which act via a
distinct domain, referred to as the ``P''-site from a
requirement for a purine
moiety(1, 2, 3, 4, 5, 6, 7, 8) .
Of the mammalian adenylyl cyclases that have been tested, all save the
enzyme from sperm have been found to be susceptible to P-site-mediated
inhibition(5, 9, 10, 11, 12) .
Although P-site ligands act directly on adenylyl cyclase, enzyme
stimulated by hormones (via G
) or Mn are most sensitive to inhibition, and P-site ligands may be
viewed as attenuating the enzyme's susceptibility to such
activation(5, 8, 13, 14, 15, 16, 17, 18) .
)Shared key
structural requirements for substrate and for P-site ligands include a
requirement for adenine, enhanced efficacy with 2`-deoxyadenosine
derivatives compared with those of adenosine, and a requirement
(catalysis) or preference (inhibition) for
phosphate(1, 2, 3, 4, 5) .
Given the shared requirements of these ligands, some similarities in
the binding domains for each may be expected. Lacking have been high
affinity labeled ligands suitable for binding to either or both
domains.
)and the naturally occurring 3`-AMP and 2`-d-3`-AMP, with
IC values in the micromolar
range(5, 22) . In the synthesis of 2`,5`-dd-3`-AMP,
referred to in a previous study(5) , a small amount of a more
potent inhibitory peak also was detected in HPLC eluates. Upon ashing,
this unexpected inhibitor was found to contain two phosphates per
adenine (23) and the most likely product was 2`,5`-dd-3`-ADP.
Subsequently, we developed a synthesis for the 3`-polyphosphorylated
derivatives of 2`,5`-ddAdo(24) . Reported here are the effects
of these agents on adenylyl cyclase and the mode of their inhibition.
Preparation and Assay of Adenylyl
Cyclase
Detergent-solubilized preparations of adenylyl cyclase
from rat and bovine brains were prepared and assayed as described
previously(5, 14, 15) . Bovine brain adenylyl
cyclase was purified as described by Pfeuffer et
al.(25) . Inhibition kinetics were determined on enzyme
assayed with concentrations of divalent cation fixed in excess of the
ATP concentration as described previously(26) . Inhibition was
determined on enzyme that was activated in the presence of
Mn and forskolin.
Estimation of Bound Phosphate
Bound phosphate was
determined on ashed samples by the malachite green method of Stull and
Buss(23) . Adenine was estimated by uv-absorption at 259 nm
following purification on high performance liquid chromatography.Materials
2`,5`-dd-3`-ADP and 2`,5`-dd-3`-ATP were
prepared as described previously(24) . Lubrol PX (from Sigma)
was filtered through alumina (Neutral, AG7, from Bio-Rad Laboratories)
to remove peroxides. Recombinant Type I enzyme was generously supplied
by Drs. R. Taussig and A. G. Gilman. Enzyme was purified by forskolin
affinity chromatography from extracts of fall army worm ovarian (Sf9)
cells infected with a Type I adenylyl cyclase encoding
baculovirus(27) . Other reagents were of the highest quality
from commercial sources.
values for
inhibition of the enzyme by these nucleotides are presented in Table 1and are compared with those of the parent nucleoside and
with known ligands. 2`,5`-dd-3`-ADP and 2`,5`-dd-3`-ATP exhibited
IC
values of 0.1 µM and 0.04 µM,
respectively. Potency of the 3`-monophosphate (2`,5`-dd-3`-AMP,
IC
0.5 µM) was enhanced approximately
5-fold by the addition of the second phosphate at the 3`-position
(2`,5`-dd-3`-ADP) and an additional two-plus-fold by the addition of
the third phosphate (2`,5`-dd-3`-ATP). Thus, the most effective
inhibitory ligands of adenylyl cyclase are effected by the removal of
both 2`- and 5`-hydroxyl groups from adenosine and the addition of
polyphosphate at the 3`-position (Table 1). The rank order noted
here was maintained with the purified native bovine type I and with the
recombinant wild type type I adenylyl cyclases, although with these
enzymes each of the 3`-nucleotides was noticeably less potent than with
the enzyme in the cruder detergent-dispersed rat brain preparation (Table 2). The reason for the loss of inhibitory potency of these
3`-nucleotides upon purification of adenylyl cyclase is unknown but is
consistent with previously noted observations with 2`-d-3`-AMP and
2`,5`-ddAdo with the bovine brain enzyme(5) . The reduced
sensitivity may be due simply to the changes in structure of the enzyme
upon isolation and removal of the native phospholipid environment.
, 100 µM 5`-ATP, 1 mM 3-isobutyl-1-methylxanthine, 100 µM forskolin, 1 mg
of bovine serum albumin per ml, 3 mM dithiothreitol, 0.1%
(w/v) Lubrol PX, 50 mM MOPS buffer, and an ATP-regenerating
system including 4 mM phosphoenolpyruvate and 100 µg of
pyruvate kinase per ml.
40 nM) is almost two
orders of magnitude more potent than previous ligands in inhibiting
native adenylyl cyclase. It the most potent non-protein regulator of
adenylyl cyclases thus far described. Since inhibition was also
observed with purified adenylyl cyclase, it is clear that
2`,5`-dd-3`-ATP acts directly on the enzyme, exerting inhibition
independent of either hormone receptor, stimulatory or inhibitory
G-protein, or G-protein subunit. It approaches the potency of the
stimulatory effect of rG
on the type I adenylyl
cyclase and the stimulatory and inhibitory effects of 
on
-activated types II and I, respectively(27) . 
ATP or MgATP (1, 3, 7,
8, 13, 14, 16, 28). Thus, the evidence supports the conclusion that
adenylyl cyclases contain distinct and interacting adenine nucleotide
binding domains for catalysis (5`-ATP) and inhibition
(2`,5`-dd-3`-ATP).
in excess of the ATP
concentration, 100 µM forskolin, and an ATP-regenerating
system including 2 mM creatine phosphate and 100 µg of
creatine kinase per ml.
))
We are grateful to Drs. R. Taussig and A. G. Gilman
for providing the recombinant type I adenylyl cyclase used in some of
the experiments referred to here.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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