(Received for publication, April 15, 1997, and in revised form, June 20, 1997)
From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041
Fragments of the two cytoplasmic domains of
mammalian adenylyl cyclases can be synthesized independently (and
abundantly) as soluble proteins; Gs- and
forskolin-stimulated enzymatic activity is restored upon their mixture.
We have utilized this system to characterize the interactions of
adenylyl cyclase with forskolin and its substrate, ATP. In the presence
of Gs
, adenylyl cyclase is activated in response to
occupation of only one forskolin-binding site. A single binding site
for forskolin was identified by equilibrium dialysis; its
Kd (0.1 µM) corresponds to the
EC50 for enzyme activation. The affinity of forskolin for
adenylyl cyclase is greatly reduced in the absence of Gs
(~40 µM). Binding of forskolin to the individual
cytoplasmic domains of the enzyme was not detected. A single binding
site for the ATP analog,
,
-methylene ATP (Ap(CH2)pp),
was also detected by equilibrium dialysis. Such binding was not
observed with the individual domains. Binding of Ap(CH2)pp
was unaffected by P-site inhibitors of adenylyl cyclase. A modified
P-loop sequence located near the carboxyl terminus of adenylyl cyclase
has been implicated in ATP binding. Mutation of the conserved,
non-glycine residues within this region caused no significant changes
in the Km for ATP or the Ki for
Ap(CH2)pp. It thus seems unlikely that this region is part
of the active site. However, a mutation in the C1 domain
(E518A) causes a 10-fold decrease in the binding affinity for
Ap(CH2)pp. This residue and the active site of the enzyme
may lie at the interface between the two cytosolic domains.
Mammalian adenylyl cyclases have a characteristic membrane-bound
topology, consisting of a short, intracellular amino terminus, followed
by a set of six transmembrane spans, a large (~40 kDa) cytoplasmic
domain (designated C1), a second set of six transmembrane spans, and a carboxyl-terminal cytoplasmic domain (C2),
which is homologous to C1 (1, 2). However, very little
direct structural information has been available until recently, and there is no knowledge of the number and nature of catalytic sites, as
well as those for a plethora of activators and inhibitors. The
elimination of several of the proteins' domains, including the amino
terminus and the transmembrane spans, has permitted expression of a
soluble version of mammalian adenylyl cyclase (3, 4). This molecule
displays many of the interesting regulatory features that characterize
these enzymes: synergistic interactions between activators
(e.g. between
Gs1 and
forskolin) and inhibition by P-site inhibitors. Remarkably, these
important regulatory properties and highly efficient catalysis are both
displayed by a mixture of two independently synthesized ~25-kDa
fragments of adenylyl cyclase, one each from C1 and
C2. Both of these small proteins can be synthesized and
purified in quantities sufficient to pose basic biochemical and
structural questions (5-7).
All mammalian adenylyl cyclases are activated by forskolin, a naturally
occurring diterpene (8). Forskolin binds to several intrinsic membrane
proteins, including adenylyl cyclases, glucose transporters,
voltage-gated K+ channels, ligand-gated ion channels, and P
glycoproteins (9). It was believed that this hydrophobic molecule bound
in or near the membrane spans, as it appears to do with P glycoproteins
and glucose transporters (10, 11). However, construction and expression of the soluble mammalian adenylyl cyclase mentioned above permitted unequivocal demonstration that the capacity of this enzyme to be
activated by forskolin resides within its cytoplasmic domains. Although
there is no evidence that a forskolin-like molecule plays a
physiological role in regulation of adenylyl cyclase activity, investigation of its interactions with the enzyme will contribute to
comprehension of mechanisms of stimulation and synergistic regulation
of catalytic activity. To date, we know that forskolin increases the
apparent affinity of the C1 and C2 domains of
adenylyl cyclase for each other and promotes much more efficient
catalysis as a result of this protein-protein interaction; the same can be said of Gs (5, 6).
Mystery also shrouds the ATP-binding site(s) of adenylyl cyclase. Devoid of any traditional P-loop or Walker motifs (1, 12), the number and location of catalytic sites in the molecule are unknown, although its tandemly duplicated structure provides grounds for speculation. To date, we have demonstrated, not surprisingly, that only one molecule of ATP is consumed during the synthesis of one molecule of cyclic AMP and one molecule of pyrophosphate (4). In addition, all mammalian adenylyl cyclases are inhibited by adenosine and certain analogs thereof (13-15). The mechanism and location of this inhibitory site (the so-called P site) and its relationship to the substrate-binding site are also unknown.
We are now able to characterize the interactions of both forskolin and a nonsubstrate analog of ATP (Ap(CH2)pp) with the cytosolic, catalytic domains of mammalian adenylyl cyclase in a reasonably rigorous and quantitative manner. The results of these studies are described below, as are analyses of mutants that provide insight into the location of the catalytic site.
[-32P]ATP (800 Ci/mmol),
[12-3H]forskolin (31 Ci/mmol), and Ap(CH2)pp
(20 Ci/mmol) were purchased from NEN Life Science Products. All
tritiated compounds were lyophilized regularly to remove
[3H]H2O. Unlabeled Ap(CH2)pp
was purchased from ICN.
Recombinant Gs,
IIC2, and VC1(591)Flag were purified from
Escherichia coli as described (5, 7, 16). Recombinant Gs
was activated by incubation with 50 mM
NaHepes (pH 8.0), 10 mM MgSO4, 1 mM
EDTA, 2 mM dithiothreitol, and 400 µM GTP
S at 30 °C for 30 min. Free GTP
S was removed by gel filtration. GTP
S-activated Gs
was utilized in all experiments,
and the active protein concentration was determined by quantification of GTP
S binding. Preparations of Gs
utilized for
titrations (Figs. 1 and 2) were 90% active. Gs
was
present in excess for other experiments and was typically 75%
active.
Mutagenesis of IIC2 and VC1
IIC2 was subcloned into the pAlter-1
mutagenesis vector (Promega) by digestion of
pQE60-IIC2H6 (5) with the restriction enzymes
EcoRI and HindIII. VC1 was subcloned
by digestion of pQE60-H6-VC1(364-591)Flag (7)
with NcoI and HindIII and utilizing the vector,
pAlter-IC1IIC2L3, which contained
an NcoI site at the extreme 5 end of the
IC1IIC2L3 clone (4). Mutations of
IIC2 residues Tyr-1054, Arg-1059, and Lys-1065 to alanine
were created using the oligonucleotides
5
-GCAGACGCTTGGCGCCACGTGTACATGTC, 5
-CACGTGTACATGTGCAGGTATCATCAATGTG,
and 5
-GGTATCATCAATGTGGCGGGGAAAGGGGAC, respectively. Mutation of
VC1 residue Glu-518 to alanine was accomplished with the
oligonucleotide 5
-GGCCAACCATATGGCAGCTGGAGGCAAG. Amino acids are
designated according to their position in the full-length, membrane-bound adenylyl cyclases. The mutations R1059A and Y1054A of
IIC2 were returned to the expression plasmid
pQE60-IIC2H6 using the restriction sites
BsrGI and HindIII. Mutant K1065A was cloned with
the restriction enzymes BstXI and HindIII. The
mutation E518A of VC1 was placed in its appropriate
expression vector with the restriction enzymes NcoI and
HindIII. Each mutant cDNA was sequenced and expressed in
E. coli strain BL21(DE3).
Assays were performed as
described (17) for 10-15 min at 30 °C in a final volume of 100 µl, unless stated otherwise. The final concentrations of free
MgCl2 and MnCl2 were 10 and 1.8 mM, respectively. Activities are expressed per milligrams of the limiting adenylyl cyclase domain in the assay (VC1 or
IIC2). For determination of kinetic constants, MgATP or
MnATP was varied from 20 µM to 2.56 mM with a
fixed excess of Mg2+ or Mn2+. Titrations of
Gs or forskolin were performed using a high but limiting
concentration of one cyclase domain (1.5-2 µM) and an
excess of the second (2.9-3 µM). These assays were
carried out at 4 °C (to slow catalysis at high protein
concentrations) in a final volume of 50 µl. Reactions in which
Gs
was titrated contained 100 µM soluble
form of forskolin,
6-O-[3
-(piperidino)propionyl]forskolin (Calbiochem),
and serial dilutions of Gs
from 31 nM to 6 µM (final concentration). Reactions in which forskolin
was titrated contained 3 µM Gs
and serial
dilutions of forskolin as indicated, yielding a final concentration of
0.4% ethanol in the assay. Proteins and forskolin were first incubated
for 6 min on ice before initiation of the reaction with 10 mM [
-32P]ATP. Reactions were terminated
after 5 min with 850 µl of stop solution containing 0.25% sodium
dodecyl sulfate, 5 mM ATP, and 0.175 mM cyclic
AMP. [32P]cAMP was isolated by sequential chromatography
on Dowex-50 and Al2O3; [3H]cAMP
was added to monitor recovery of [32P]cAMP during
purification.
Equilibrium dialysis chambers were
purchased from Hoeffer. Chambers (60 µl) were separated by dialysis
membrane with a cutoff of 14 kDa. To examine forskolin binding, each
chamber contained 20 mM NaHepes (pH 8.0), 10 mM
MgCl2, 1 mM dithiothreitol, and the indicated
amount of [3H]forskolin (0.05-5 µM). One
chamber contained all three proteins required for binding: 2 µM VC1, 4 µM IIC2,
and 6 µM Gs-GTP
S; the opposite chamber
contained either buffer alone or only 5 µM IIC2 (or 4 µM VC1) and 6 µM GTP
S-Gs
to minimize the effects of
any nonspecific binding. Similar results were obtained with both
methods. For measurements in the absence of GTP
S-Gs
, one chamber contained 30 µM VC1 and 120 µM IIC2, while the opposite chamber contained
either buffer alone or 120 µM IIC2. Samples were removed after dialysis for 24 h at 4 °C with rotation;
duplicate 15-µl aliquots from each chamber were analyzed by liquid
scintillation spectrometry.
Equilibrium dialysis reactions with the ligand Ap(CH2)pp
contained 20 mM NaHepes (pH 8.0), 0.65 mM
MnCl2, 0.1 mM ascorbate, and the indicated
amount of [3H]Ap(CH2)pp (1-40
µM). One chamber included 30 µM each of
VC1, IIC2, and GTPS-Gs
.
Samples were processed as just described.
To determine the number of
binding sites for forskolin and substrate in a complex of
VC1 and IIC2, it is essential to know the
fraction of protein in each of the two preparations that is active,
i.e. capable of forming a catalytically productive complex with its partner. We know that Gs, VC1, and
IIC2 form a complex containing one molecule of each protein
(7), and the fraction of purified Gs
that is native is
readily measured by assessing radioactive guanine nucleotide binding.
We thus titrated mixtures of VC1 and IIC2 with
Gs
or forskolin, observing the increase in activity
caused by small incremental additions of activator. To obtain precise
information, these experiments must be performed at concentrations well
above the Kd for the interaction of the two domains
of the enzyme and for the interaction of the activator with the enzyme.
Thus, high concentrations of forskolin were included for titrations
with Gs
and vice versa. Experiments were performed with
either a very high concentration of VC1 in excess of a high
concentration of IIC2 (1.5 µM) (Fig.
1); or with a very high concentration of
IIC2 in excess of a high concentration of VC1
(2 µM) (Fig. 2). Titrations
with Gs
(with either IIC2 (Fig.
1A) or VC1 (Fig. 2A) limiting)
indicated that 80 and 75% of the protein in the preparations of
IIC2 and VC1, respectively, was functional.
Titrations with forskolin, performed with the same protein mixtures and
at the same time, demonstrated that essentially identical amounts of
forskolin and Gs
were required to achieve maximal
activity, indicating that only one molecule of forskolin is necessary
for maximal enzyme activation (Figs. 1B and
2B).
We have quantified
forskolin binding by equilibrium dialysis. Binding of the diterpene to
either VC1 (40 µM) or IIC2 (120 µM) alone is not observed over the range of
concentrations that could be explored (in the presence or absence of
Gs). Successful experiments were performed at limiting
concentrations of one of the cyclase domains and excess concentrations
of the second to ensure maximal formation of
VC1/IIC2 heterodimers (Fig.
3, A and B). The
concentrations of the adenylyl cyclase domains required for association
in the presence of Gs
are relatively low (~2-4
µM). Regardless of which domain was limiting, it is clear that 1 molecule of forskolin binds to a C1/C2
heterodimer. The Kd for forskolin binding, 0.09 ± 0.03 µM (n = 6), is very similar to
the EC50 for activation of the enzyme (0.1 µM) (7) (data not shown). We were not successful in
attempts to detect a second binding site for forskolin using much
higher protein concentrations than those used in Fig. 3. We estimate
that we would have observed a second site if its Kd
was below 60 µM.
Quantification of forskolin binding in the absence of Gs
is problematic. The EC50 for activation of the enzyme is
much greater (~15 µM), and, more importantly, the
apparent Kd of C1 for C2 is
10 µM or greater in the absence of activators. The
observed binding is shown in Fig. 4. The
estimated Kd (40 µM ± 20 µM, n = 6) compares reasonably (but not
precisely) with the EC50 for activation of
VC1/IIC2 in the absence of Gs
, 15 µM. However, saturation was not achieved because of
the insolubility of forskolin. In addition, one cyclase domain
(C2) was present at very high concentrations (120 µM) to drive the interactions, and the proteins are less
stable in the absence of additional activators.
Inhibition of Adenylyl Cyclase by Ap(CH2)pp
The
ATP analog Ap(CH2)pp is a competitive inhibitor of adenylyl
cyclase and is not utilized by the enzyme as a substrate. The
IC50 for Ap(CH2)pp is dependent on the divalent
cation utilized. In the presence of Gs and
Mg2+, the Ki for Ap(CH2)pp
is equal to the Km for ATP (320 µM).
However, in the presence of Mn2+, the Ki
for Ap(CH2)pp is 15 µM, roughly 10-fold lower than the Km
for ATP under these conditions (Fig. 5; Table I). Similar phenomena are observed when
forskolin is the activator (Table I). These observations do not appear
to be due to simple structural differences between ATP and
Ap(CH2)pp, since the Ki for
Ap(CH2)pp is the same as the Km for ATP
in the presence of Mg2+.
|
Analysis of
binding of [3H]Ap(CH2)pp to adenylyl cyclase
by equilibrium dialysis in the presence of Gs and
Mn2+ revealed a single binding site per
C1/C2 heterodimer with a Kd of 7 ± 0.5 µM (n = 3), similar to
the value of Ki under these conditions (15 µM) (Fig. 6). There is no
detectable binding of [3H]Ap(CH2)pp to either
VC1 or IIC2 alone (data not shown).
Adenylyl cyclases are inhibited by direct interactions with adenosine
analogs known as P (purine)-site inhibitors. This phenomenon is curious
kinetically, since inhibition of VC1/IIC2 is
uncompetitive with respect to MgATP and noncompetitive with respect to
MnATP. A potent P-site inhibitor, 2-deoxy-3
-AMP, was tested for its capacity to disrupt binding of Ap(CH2)pp. Although the
Ki for 2
-deoxy-3
-AMP is 7 µM in the
presence of Gs
and Mn2+, binding of
[3H]Ap(CH2)pp was unaffected by 400 µM 2
-deoxy-3
-AMP (Fig. 6). This prototypical P-site
inhibitor clearly does not compete for binding of ATP to the free
enzyme or disrupt ATP binding in any capacity.
Droste et al.
(18) identified a 25-amino acid peptide near the carboxyl terminus of
type I adenylyl cyclase (residues 1027-1051) that interacted with the
photoaffinity probe [-32P]8-N3ATP. We have
mutated each of the conserved, non-glycine residues within this region
of IIC2 to alanine and measured the resultant effects on
the Km for ATP, Vmax, and
Ki for Ap(CH2)pp in the presence of
VC1 (Table II). All of the
mutant proteins displayed synergistic activation in the presence of
Gs
and forskolin, suggesting that there was no gross
disruption of structure. The yields for two of the mutant proteins,
K1065A and Y1054A, were poor, and they displayed greatly reduced
activity. However, changes in Km and
Ki values were modest in the presence of
Mg2+, and these values were normal with Mn2+.
The yield of mutant R1059A was normal, as was its
Vmax. Interestingly, its Km
for ATP and Ki for Ap(CH2)pp were
elevated 10- and 3-fold, respectively, with Mg2+, but these
values were identical to those for the wild type protein in the
presence of Mn2+. Equilibrium dialysis studies with this
protein demonstrate a normal Kd for
Ap(CH2)pp binding in the presence of Gs
and
Mn2+. The only other conserved residue in this sequence is
Lys-1067, which was previously mutated to alanine in type I adenylyl
cyclase (19); there was no effect on Vmax or
Km for ATP. It thus seems unlikely that this region
is a component of the substrate binding site.
|
Amino acid residue Glu-432 of type I adenylyl cyclase (analogous to
Glu-518 of VC1) was described previously as a
Km mutant (19) and provided the opportunity to see
if its affinity for substrate is altered. The yield of the
VC1-E518A mutant protein was reasonable, but it had low
activity in the presence of Mg2+
(Vmax = 0.7 µmol/min·mg). Its
Km for ATP and Ki for
Ap(CH2)pp were elevated approximately 10-fold when
Mg2+ was present (Table II). In the presence of
Mn2+, Vmax was elevated more than
200-fold (compared with Mg2+) to exceed wild type values,
while the Km and Ki remained
abnormally high. This mutant protein is activated synergistically by
Gs and forskolin, and its affinity for Gs
is altered by no more than 2-fold. The apparent affinity of this mutant
C1 domain for IIC2 is also only slightly
altered (Fig. 7). This is an important
point, since the newly described crystal structure of a
IIC2 homodimer suggests that this residue lies at the
interface between the two domains (20).
The interaction of forskolin with soluble complexes of the
cytosolic domains of adenylyl cyclase has been demonstrated by two
independent methods. In the presence of Gs, only one forskolin molecule is required to activate the enzyme (Figs. 1 and 2)
and only one binding site can be detected (Fig. 3); the Kd for this site corresponds to the EC50
for activation. Lower affinity sites were not detected at high
concentrations of enzyme. In the absence of Gs
,
forskolin binds to a low affinity site with a Kd of
approximately 40 µM, a value that is somewhat higher than
the EC50 for enzyme activation under these conditions (15 µM). Although these latter experiments were technically
difficult, this discrepancy might suggest that the binding curve (Fig.
4) reflects a mixture of contributions from nonidentical sites. If
true, the hypothetical second (lowest affinity) site has no apparent
effect on enzymatic activity.
Others have suggested the possibility of two binding sites for
forskolin. Using membrane-bound type I adenylyl cyclase, Sutkowski et al. (21) identified two apparent binding sites for an
isothiocyanate derivative of the diterpene. The high affinity (300 nM) site appeared to be silent, in that covalent binding of
the derivative to this site inhibited forskolin binding at
concentrations significantly lower than those required to activate the
enzyme. Occupation of a low affinity (10 µM) site caused
a concentration-dependent loss of forskolin-stimulated
adenylyl cyclase activity (although other activators were not tested).
It is notable that type I adenylyl cyclase is activated independently,
not synergistically, by Gs and forskolin. Zhang et
al. (20) have recently solved the crystal structure of a homodimer
of IIC2. This assembly includes two forskolin molecules
bound at the interface between the monomers. It seems likely that the
overall structure of the C2 homodimer will resemble that of
a C1/C2 heterodimer.
To us, the most straightforward model to rationalize these observations
(for enzymes displaying synergistic activation by Gs and
forskolin) is that there exists a single binding site for forskolin
that is responsible for enzyme activation. This site has relatively
high affinity for the diterpene in the presence of Gs
and relatively low affinity for forskolin in the absence of
Gs
. This does not preclude the existence of another very low affinity site that is not coupled to changes in catalytic activity.
Such sites might well be filled during protein crystallization, performed at very high concentrations of protein (20 mg/ml) and forskolin (1 mM) (20). An alternative explanation (again
for enzymes that display synergy between forskolin and
Gs
) is that we have observed two distinct
forskolin-binding sites: a high affinity site that is occupied only in
the presence of Gs
and a separate, low affinity site
that is occupied only in its absence; occupation of either site
activates the enzyme. The high affinity site observed by Sutkowski
et al. (21), which is not linked to changes in enzymatic
activity, might be characteristic of enzymes that do not display
synergistic activation by forskolin. This site may be quite distinct
from those that regulate catalysis and/or may reflect binding
properties of the complete, membrane-bound enzyme. The low affinity
site on type I adenylyl cyclase resembles that observed on the soluble
VC1-IIC2 complex in the absence of Gs
.
The homologous nature of the C1 and C2 domains
suggests the possibility of two nucleotide-binding sites, as occur in P
glycoproteins (22) and cystic fibrosis transmembrane conductance
regulator (23). Alternatively, a single ATP-binding site may be wholly contained within one domain or shared by the two domains at their interface. If there is a multiplicity of catalytic sites, the pure
hyperbolic nature of plots of reaction velocity versus ATP concentration demands their near identity and a lack of interaction between them. Using equilibrium dialysis and a non-substrate ATP analog, Ap(CH2)pp, we have identified a single ATP-binding
site. The affinity of nucleotide for this site is greatly influenced by
the metal cofactor, as shown by the large difference in the Ki for Ap(CH2)pp in the presence of
Mg2+ and Mn2+. Gs and forskolin
do not have substantial effects on the Ki for
Ap(CH2)pp, suggesting that these activators do not
influence substrate binding significantly. In the case of MnATP, the
Km for ATP is only partially determined by substrate
binding, as indicated by the difference between the Kd for MnAp(CH2)pp and the
Km for ATP; this difference is inapparent when
Mg2+ replaces Mn2+.
All mammalian adenylyl cyclases are inhibited directly by adenosine analogs known as P-site inhibitors (24), which display several unique features. Stimulated forms of the enzyme are substantially more sensitive to P-site inhibition than are nonactivated forms (13-15, 25). Inhibition is dependent on metal and is characteristically uncompetitive or noncompetitive with respect to metal-ATP, depending on the activator and the metal (26). A large excess of a potent P-site inhibitor had no effect on either the amount of Ap(CH2)pp bound or its affinity, as measured by equilibrium dialysis (Fig. 6). This result provides important clues to the mechanism of P-site inhibition, suggesting that the inhibitor must either bind at an intermediate point along the reaction coordinate or to an entirely separate site on the enzyme that does not mediate its inhibitory effects via alterations of substrate binding. Florio (27) and Florio and Ross (25) suggested the possibility of dead-end inhibition through the formation of an adenylyl cyclase-pyrophosphate-inhibitor complex that would include both pyrophosphate and a P-site inhibitor at the active site. Johnson and Shoshani (26) have proposed a distinct binding site for P-site inhibitors in which both substrate and inhibitor are bound simultaneously (26). Both models are supported by a great deal of kinetic data, and there is no current means to distinguish between the two.
Adenylyl cyclase lacks the conventional motif GXXXXGKS,
characteristic of nucleotide-binding sites, called the
phosphate-binding or P-loop (12). However, an alternative sequence,
G(X0-7)KG(X0-4)L(X0-5)S/T, is conserved in mammalian, bacterial, and yeast adenylyl cyclases (28).
A 25-amino acid peptide, located near the carboxyl terminus of type I
adenylyl cyclase and overlapping with the modified consensus site, was
identified as the labeling site for the photoactivatable ATP analog,
[-32P]8-N3ATP (18). We have mutated each
of the conserved, non-glycine amino acid residues within the peptide
region; none of these proteins exhibits any appreciable difference in
its Km for ATP or its Ki for
Ap(CH2)pp. Mutation of the key lysine residue in the P-loop
of p21ras or adenylate kinase leads to a reduction in substrate
binding of 100- or 20-fold, respectively, along with variable decreases in kcat (29, 30). It thus seems unlikely that
Lys-1065 (or Lys-1067, examined in Ref. 19) plays a role in substrate
binding. Although no change in binding was observed, there was an
appreciable change in Vmax for 2 of these
mutants (Y1054A and K1065A). Levin and Reed (31) identified this region
as necessary for regulation of activity by protein kinase C. Mutation
of this regulatory region may thus change catalytic activity.
Since neither isolated cytosolic domain of adenylyl cyclase can bind Ap(CH2)pp, the interface of these two molecules is a logical spot for the active site. However, identification of this site has been difficult, and few Km mutants of adenylyl cyclase have been identified. Of the 19 conserved residues that have been mutated to produce adenylyl cyclases that retain catalytic activity (Refs. 19 and 20, and this study), only 2 show any significant change in Km when assayed in the presence of Mn2+. One of these (E518A of type V) is located in the C1 domain and has a 10-fold reduction in its Km for ATP. We have shown that this reduction in Km is reflected in a reduction of substrate binding. Assuming a similar fold for both C1 and C2, Glu-518 is located near the interface of the two domains (20). The altered residue in the only other Km mutant of adenylyl cyclase that has been identified (K923A in type I) and several other residues that are essential for catalysis are located in the C2 domain but also at the interface between C1 and C2 (19, 20). Thus, the nucleotide-binding motif of adenylyl cyclase appears to differ greatly from the traditional motifs of G proteins, kinases, and other ATP-requiring enzymes. We presume that residues lining the interface between the two domains contribute to binding and catalysis. This arrangement may permit optimal linkage of small changes in conformation to large changes in catalytic activity.
We thank Jeff Laidlaw for excellent technical assistance and Arnold E. Ruoho for providing information about the structure of the IIC2 homodimer prior to publication.