From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0636
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The recent determination of the crystal structure of adenylyl cyclase has elucidated many structural features that determine the regulatory properties of the enzyme. In addition, the characterization of adenylyl cyclase by mutagenic techniques and the identification of the binding site for P-site inhibitors have led to modeling studies that describe the ATP-binding site. Despite these advances, the catalytic mechanism of adenylyl cyclase remains uncertain, especially with respect to the role that magnesium ions may play in this process. We have identified four mutant mammalian adenylyl cyclases defective in their metal dependence, allowing us to further characterize the function of metal ions in the catalytic mechanism of this enzyme. The wild-type adenylyl cyclase shows a biphasic Mg2+ dose-response curve in which the high-affinity component displays cooperativity (Hill coefficient of 1.4). Two mutations (C441R and Y442H) reduce the affinity of the adenylyl cyclase for Mg2+ dramatically without affecting the binding of MgATP, suggesting that there is a metal requirement in addition to the ATP-bound Mg2+. The results of this study thus demonstrate multiple metal requirements of adenylyl cyclase and support the existence of a Mg2+ ion essential for catalysis and distinct from the ATP-bound ion. We propose that adenylyl cyclase employs a catalytic mechanism analogous to that of DNA polymerase, in which two key magnesium ions facilitate the nucleophilic attack of the 3'-hydroxyl group and the subsequent elimination of pyrophosphate.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular levels of cAMP are primarily regulated at the level of its synthesis by adenylyl cyclase. cAMP, in turn, regulates a wide variety of cellular processes such as protein phosphorylation levels, gene expression, and ion channel conductance. Currently, nine isoforms of mammalian adenylyl cyclase have been identified by molecular cloning techniques (reviewed in Refs. 1-3), and they display a common deduced topology composed of a short cytoplasmic amino terminus followed by a region of six transmembrane domains (M1) and a large cytoplasmic loop (C1). This motif is then repeated with a second transmembrane region (M2) and a large cytoplasmic carboxyl terminus (C2). Sequence comparison has revealed that each cytoplasmic loop contains subdomains (denoted C1a and C2a) that are highly conserved among all adenylyl cyclase isoforms and that also display great homology to each other. The C1a and C2a domains can be expressed separately, and catalytic activity is reconstituted when they are mixed in vitro, although each domain by itself shows no activity (4, 5).
Many of the structural motifs of adenylyl cyclase responsible for
catalytic activity and for the recognition of regulatory molecules are
presently being uncovered. A region in the C2 domain of type II
adenylyl cyclase (residues 956-982), for example, has been implicated
in the binding of the G
protein1 subunits (6),
whereas another region in the C1 domain of type I adenylyl cyclase
(residues 495-522) appears to be involved in the regulation by
calmodulin (7, 8). The binding sites for forskolin and
Gs
, the two common activators of adenylyl cyclase, have
recently been characterized by a combination of mutagenic techniques
(9, 10) and the crystallographic analyses of the cytoplasmic domains of
adenylyl cyclase (11, 12). The crystal structures of adenylyl cyclase
also provided the basis for modeling the ATP-binding site of adenylyl
cyclase (12, 13); however, these structures do not include the MgATP
substrate, and several questions remain about the precise catalytic
mechanism of this enzyme.
The structure of adenylyl cyclase was observed to be remarkably similar
to that of DNA polymerase (14), despite low sequence homologies. This
similarity is not surprising because both enzymes catalyze analogous
chemical reactions, involving the in-line attack of a ribose
3'-hydroxyl on the -phosphate of a nucleotide triphosphate, resulting in the release of a pyrophosphate group. DNA polymerase employs two key catalytic aspartate residues that bind two essential magnesium ions (15-17). One Mg2+ion is associated with the
nucleotide triphosphate, whereas the second one coordinates the
3'-hydroxyl, thus facilitating the nucleophilic attack. The positions
of these aspartate residues are spatially conserved in the structure of
adenylyl cyclase, where they were shown to bind to at least one
Mg2+ ion (12), suggesting that the catalytic mechanism
employed by both enzymes is also conserved. Modeling studies on the
active site of adenylyl cyclase have predicted a single ATP-bound
magnesium ion (12, 13) and suggest that the 3'-hydroxyl is deprotonated by an acidic residue. Although the crystal structure of adenylyl cyclase shows only a single Mg2+ ion (12), an alternative
model in which a second Mg2+ ion promotes this
deprotonation was acknowledged. Thus, the exact role and number of
metal ions in the adenylyl cyclase-catalyzed reaction remain to be
elucidated.
We have employed a genetic selection system for the identification of mutant adenylyl cyclases defective in their regulatory properties (10). This system relies on the expression of mammalian adenylyl cyclase in yeast (Saccharomyces cerevisiae) and makes use of the requirement of cAMP for growth in yeast (18). In this manner, we identified four mutant mammalian adenylyl cyclases displaying specific defects in their metal dependence. These mutations occur in highly conserved residues that are located in proximity to each other and to the two aspartate residues that coordinate magnesium. Characterization of the metal dependence of these mutants has shown that two of the mutations (C441R and Y442H) cause severe catalytic defects when assayed in the presence of magnesium, without compromising the ability of adenylyl cyclase to bind MgATP. Our data provide evidence for the existence of multiple magnesium-binding sites and strongly support a two-metal mechanism in which one metal ion is complexed with ATP, whereas the second plays a primarily catalytic role. Such a mechanism is remarkably similar to that identified for DNA polymerase, and we propose that this catalytic mechanism may be shared by a diverse family of enzymes involved in phosphoryl transfer reactions.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Strains, Plasmid Construction, and Mutant
Selection--
Yeast strain TC41-1 (19) (MATa,
leu2-3, leu2-112, ura3-52, his3, his4, cam1, cam2, cam3,
cyr1 ::URA3) was a generous gift of Warren
Heideman (University of Wisconsin, Madison, WI). The construction of an
isogenic derivative of this strain expressing rat Gs
and
the construction of the plasmid pADHprACVLeu encoding the dog type V
adenylyl cyclase (20) were previously described (10). Randomly mutated
libraries of plasmid pADHprACVLeu were generated by error-generating
polymerase chain reaction mutagenic techniques as described elsewhere
(10). Both the selection of type V adenylyl cyclase mutants and the
retesting of these mutants in yeast were performed as described
previously (10).
DNA Sequencing-- Sequencing of the adenylyl cyclase mutants was performed using Thermal Sequenase (Amersham Pharmacia Biotech) following the procedures supplied by the manufacturer. In some cases, automated sequencing was performed using an Applied Biosystems sequencer.
Sf9 Cell Culture and Preparation of Cell Membranes-- The procedure for the culture of Sf9 cells and the amplification of recombinant baculovirus have been outlined by Summers and Smith (21). Generation of recombinant baculovirus encoding both the wild-type and mutant type V adenylyl cyclases was performed using the BAC-to-BAC system (Life Technologies, Inc.) as described previously (10). Sf9 membranes containing individual adenylyl cyclase isoforms were prepared as described (22).
Purification of G Protein Subunits--
Recombinant
Gs was synthesized in bacteria and purified as described
by Lee et al. (23). Protein concentrations were estimated by
staining with Amido Black (24). The
subunits were activated by
incubation with GTP
S as described previously (25).
Adenylyl Cyclase Assay-- Adenylyl cyclase activity was measured using the procedure of Smigel (26). All assays were performed for 10 min at 30 °C in a final volume of 100 µl containing 20 µg of membrane protein. The concentration of free metal ions was calculated with WinMaxC software that accounts for the buffering effects of 500 µM ATP and 400 µM EDTA in the reaction mixture.
Adenylyl Cyclase Purification and Immunoblotting-- Wild-type and mutant type V adenylyl cyclase proteins were purified from Sf9 membranes using published procedures (22). Samples were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted as described (27) using a primary rabbit antibody specific for type V/VI adenylyl cyclase (28) as described previously (10).
Data Analysis-- Data were analyzed using the GraphPad Prizm program to determine EC50 values and Hill coefficients of the dose-response curves.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously reported the development of a yeast genetic
selection system for the identification of mutant adenylyl cyclases
defective in their regulatory properties (10). Briefly, the system
relies on the expression of mammalian type V adenylyl cyclase in a
strain of yeast with a deletion in the essential CYR1 gene
locus that encodes the yeast adenylyl cyclase (18); this strain is
non-viable unless cAMP is added to the growth medium. Alternatively,
the yeast cells are able to grow when the exogenously expressed
mammalian adenylyl cyclase is activated by either forskolin or
Gs. Forskolin and Gs
activate type V
adenylyl cyclase synergistically in vitro, and the
simultaneous activation of the exogenous adenylyl cyclase in yeast by
both activators results in a synergistic increase in intracellular cAMP
levels. However, despite these high levels of cAMP, the yeast cells
fail to grow, presumably due to toxic effects of cAMP at high
concentrations. We have previously reported how this growth phenotype
was used to identify mutant adenylyl cyclases defective in their
regulation by Gs
(10).
We have employed the yeast selection system to identify adenylyl
cyclase mutants with defects in their catalytic activity. This involved
expressing a library of randomly mutagenized adenylyl cyclase
constructs in the CYR1-deleted yeast strain and selecting for transformants that grow in the presence of both forskolin and
Gs, but that have little or no growth in the presence of
either activator alone (data not
shown).2 We reasoned that the
mutations in some of these constructs may result in reduced catalytic
activities while retaining sufficient activity in the presence of
synergistic activation by Gs
and forskolin to support
growth.
To determine whether the biochemical properties of the mutants could
account for the altered growth phenotypes of the yeast, we introduced
the mutant constructs into recombinant baculovirus and overexpressed
them in Sf9 cells; membranes from these cells were then used as
a source of adenylyl cyclase for biochemical studies. Initial
characterization involved measuring the activity of the wild-type and
mutant enzymes in response to various activators in the presence or
absence of Mn2+. For wild-type type V adenylyl cyclase, the
addition of Mn2+ to adenylyl cyclase resulted in an
enhanced basal and forskolin-stimulated catalytic activity, in
agreement with previously published studies (29), with little or no
effect on the Gs-stimulated enzymatic activity (Fig.
1A). Four mutants (F423L,
R434S, C441R, and Y442H) were identified that have common defects in
their catalytic activity, displaying low activities in the presence of
Mg2+, while showing normal activities when Mn2+
was added (behavior exemplified by the C441R mutant) (Fig.
1B). This suggests that these mutants are not catalytically
compromised, but instead display specific defects in their metal
dependence.
|
Recent advances in the structural characterization of adenylyl cyclase
have suggested different roles for metal ions in the catalytic
mechanism of adenylyl cyclase. The determination of the crystal
structure of the catalytic core of adenylyl cyclase has led to models
predicting a single Mg2+ ion present in the MgATP substrate
complex (12, 13). These structural data, however, did not elucidate how
the 3'-hydroxyl is deprotonated, and the possibility that a second
Mg2+ ion could assume this role was not excluded (12). The
structure of the adenylyl cyclase catalytic core was shown to be highly similar to that of the DNA polymerase palm domain (14). DNA polymerase
employs two magnesium ions as part of its catalytic mechanism; one is
associated with the - and
-phosphates of the incoming nucleotide
triphosphate, whereas the other coordinates to and activates the
attacking ribose 3'-hydroxyl of the terminal nucleotide (reviewed by
Steitz (17)). The homology of the structures of adenylyl cyclase and
DNA polymerase and the similarities in the chemical reactions they
catalyze have led to the prediction that these enzymes share the same
catalytic mechanism.
To elucidate the role of metal ions in the catalytic mechanism of
adenylyl cyclase, we characterized the metal dependence of the
wild-type and mutant enzymes by measuring enzymatic activity at varying
levels of Mg2+ or Mn2+ (Fig.
2). As shown in Fig. 2A, the
forskolin-stimulated wild-type adenylyl cyclase displayed a biphasic
dose-response curve with a high-affinity component (EC50 ~ 400 µM) and a low-affinity component that only became
apparent at non-physiological concentrations of free Mg2+
(>10 mM). In contrast, we only observed a single magnesium
component for the Gs-stimulated adenylyl cyclase
(EC50 ~ 300 µM), and concentrations of
Mg2+ above 2 mM actually inhibited this
activity (data not shown).
|
All four adenylyl cyclase mutants displayed a rightward shift in their
Mg2+ dose-response curves (Fig. 2, A and
B), indicating a reduced affinity for the metal ion. The
mutants can be grouped according to the severity of this shift, with
the F423L and R434S mutants (group I) showing more moderate effects
(10-20-fold increase in the EC50 for Mg2+)
(Fig. 2A and Table I) and the
C441R and Y442H mutants (group II) displaying more severe shifts
(~100-fold increase in the EC50 for Mg2+)
(Fig. 2B and Table I) in their Mg2+ dependence.
All mutants displayed dramatically reduced activities when the
dose-response curve was repeated in the presence of Gs (data not shown), consistent with the observed inhibition of
Gs
-stimulated activity at the high concentrations of
Mg2+ required to observe forskolin-stimulated activity.
|
Next, we measured the activity of the wild-type and mutant adenylyl cyclases at varying concentrations of Mn2+ (Fig. 2, C and D) to evaluate the strong Mn2+ dependence of these mutants. In this case, the wild-type enzyme displayed a dose-response curve with an EC50 of 40 µM, and manganese inhibited at lower concentrations (>1 mM) than magnesium. The group I mutants with moderate Mg2+ defects (F423L and R434S) showed a Mn2+ dependence that was almost identical to that of the wild-type enzyme (Fig. 2C), with EC50 values that did not differ substantially from the wild-type value (Table I). The group II mutants (C441R and Y442H) showed an increase in the EC50 for Mn2+ (10-fold) (Table I), which paralleled the reduction in their affinity for Mg2+, although the latter effect was more severe (100-fold increase in Mg2+ EC50 versus 10-fold shift in Mn2+ EC50). Taken together, these data suggest that the disrupted metal-binding site(s) of these mutants are better able to accommodate manganese than magnesium.
The dose-response curves we observed in Fig. 2 could be due to the
formation of the metal-ATP substrate during the titration with
magnesium or manganese. However, the Mg2+ dependence of the
wild-type adenylyl cyclase did not appear to be determined solely by
the formation of MgATP because the EC50 of the
Mg2+ dose-response curve (400 µM) was
significantly higher than the Kd of ATP for
Mg2+ (64 µM). In addition, we observed little
or no catalytic activity at concentrations of Mg2+
sufficient to produce substrate (MgATP) at up to three times the
Km value of adenylyl cyclase for MgATP (80 µM) (Table I). These results are consistent with previous
reports documenting that Mg2+ concentrations in excess of
ATP are required for catalytic activity in cyc
cells of S49 murine lymphoma (30). Closer examination of the high-affinity component revealed non-hyperbolic behavior, with a Hill
coefficient of 1.4, indicating multiple Mg2+ sites (at
least two) that display cooperative binding. Our data on the wild-type
enzyme therefore suggest a role for a second Mg2+ ion in
the catalytic mechanism of adenylyl cyclase in addition to the one
present in the MgATP substrate complex.
If adenylyl cyclase utilizes only a single magnesium ion, present in the MgATP substrate complex, reducing the affinity of adenylyl cyclase for this Mg2+ should also lower the enzyme's affinity for MgATP. To evaluate this prediction, we measured the Km of the wild-type and mutant adenylyl cyclases for MgATP. As depicted in Table I, the mutants can once again be placed into two groups based on the severity of the shift in their Km values. In this case, however, the severity of the shift in the Km for MgATP was opposite from that seen in the EC50 for Mg2+. The group I mutants (F423L and R434S) displayed moderate increases both in the EC50 for Mg2+ and in the Km for MgATP, whereas the group II mutants (C441R and Y442H) displayed a dramatic increase in the EC50 for Mg2+, but showed only a minor increase in their Km for MgATP. In contrast, none of the mutants displayed significant changes in their Km for MnATP (Table I), consistent with the small effect these mutations have on enzymatic activity in the presence of Mn2+.
To confirm that the changes in Km correspond to
defects in the binding of MgATP, we measured the Ki
of these cyclase mutants for the competitive inhibitor ATPS in the
presence of Mg2+. We found that the group I mutants (F423L
and R434S) showed a large increase in the Ki for
ATP
S, whereas the group II mutants (C441R and Y442H) displayed only
a moderate reduction in their affinity for ATP
S, paralleling the
changes in Km values for MgATP (Table I). Because
the Ki values for the inhibitor are primarily
dependent on the binding affinity of the enzyme for this compound,
these data indicate that the binding of MgATP to the group II mutants
is relatively unaffected, whereas the group I mutants have a reduced
affinity for MgATP.
The large increase in the Km for MgATP and the
Ki for ATPS of F423L and R434S suggests that the
Mg2+ defect of these mutants may be primarily reducing the
affinity of adenylyl cyclase for MgATP, and we did indeed observe
normal activities when the MgATP concentrations were raised to 3 mM (data not shown). This interpretation also explains the
normal activity of the mutants in the presence of Mn2+
because the Km for MnATP is not affected by the
mutations (Table I). The dramatic reduction in the affinity of the
C441R and Y442H mutants for Mg2+, however, does not appear
to reduce the affinity of adenylyl cyclase for MgATP, suggesting that
this defect affects the binding of a Mg2+ ion that is
essential for catalysis, but not bound to ATP. It is possible that this
additional metal requirement is due to an allosteric effect, but the
similarities between adenylyl cyclase and DNA polymerase suggest that
the second Mg2+ ion fulfills the same function as the ion
coordinating the attacking 3'-hydroxyl in DNA polymerase.
We next examined the position of the mutated residues in the structure of adenylyl cyclase. All four mutations occur in the C1a cytoplasmic domain of adenylyl cyclase that is highly conserved among all isoforms of adenylyl cyclase as well as the catalytic domains of guanylyl cyclase. Sequence comparison of the nine isoforms of mammalian adenylyl cyclase and the Drosophila rutabaga gene product showed that the four mutated residues are absolutely conserved among these enzymes. Interestingly, the group II mutants are immediately adjacent to Asp-440, which is equivalent to one of two key catalytic Asp residues that bind Mg2+ in DNA polymerase (14). The function of these Asp residues appears to be at least partially conserved in adenylyl cyclase because Asp-440 and Asp-396 were also shown to coordinate to Mg2+ in the adenylyl cyclase crystal structure (12).
The crystal structure of adenylyl cyclase demonstrates that all four
mutated residues, as well as the two Mg2+-binding aspartate
residues (Asp-440 and Asp-396), occur in the same region of adenylyl
cyclase (Fig. 3). ATP and the
Mg2+ ion coordinating to the 3'-hydroxyl are not part of
the crystal structure and are depicted in their approximate positions
based on the binding sites for pyrophosphate and the P-site inhibitor 2'-deoxy-3'-adenosine monophosphate (12). Asp-440 is located at the top
of a -turn formed by the
2 and
3 strands, whereas Cys-441 and
Tyr-442 are immediately adjacent on the
3 strand. It seems likely
that mutations in these residues will result in conformational changes
of the
-turn, thus disrupting the orientation of Asp-440 and
consequently reducing the affinity of the metal-binding pocket for
Mg2+. Phe-423 and Arg-434 are more distant from these
aspartate residues and are buried beneath the adenylyl cyclase active
site. Arg-434 forms part of the
2 strand, whereas Phe-423 is located
on the preceding
2 helix. The side chains of these residues lie in
close proximity to that of Tyr-442, which points away form the
catalytic site.
|
The direct mutagenesis of either catalytic Asp residue in type I adenylyl cyclase was shown to result in inactive enzymes (13, 31) and thus does not elucidate the function of these residues in catalysis. Our functional data, however, suggest that the group II mutations (C441R and Y442H) reduce the affinity of adenylyl cyclase for metal ions by disrupting the orientation of Asp-440, confirming the essential role that this residue plays in binding Mg2+. The group I mutants (F423L and R434S), on the other hand, are more distant from the catalytic Asp residues and may affect the Mg2+-binding pocket via more global conformational changes. The Mg2+ defect of these mutants is primarily reflected in a reduced affinity for MgATP, and thus, it seems likely that the mutations may be reducing the affinity of the aspartic acid residues for the ATP-bound Mg2+. The residue equivalent to Arg-434 was previously targeted for mutagenesis in type I adenylyl cyclase (R348A in type I adenylyl cyclase) (31). In contrast to our data, the mutant shows normal activity and displays no change in the Km for MgATP, indicating that different amino acid substitutions at this position can lead to very different phenotypes. These mutagenic studies (31) also targeted Glu-518 and Arg-484 (Glu-432 and Arg-398 numbering in type I adenylyl cyclase), producing mutants that also display a strong manganese dependence. The recent availability of detailed structural information reveals that these residues are in close contact to Asp-396 (type V numbering), indicating that the effect of these mutations may be due to a disruption of the orientation of this aspartate residue. These mutants display similar phenotypes to the two groups we describe, with the E518A mutant displaying a shift in the Km for MgATP and the R484A mutant showing an enhanced manganese dependence without changes in Km. These results therefore support the role of Asp-396 in coordinating metal ions, as proposed by the crystallographic analysis of adenylyl cyclase (12).
The characterization of DNA polymerase by mutagenic techniques has also
focused on residues adjacent to the key catalytic aspartic acid
(Asp-1004 in DNA polymerase ) equivalent to Asp-440 in type V
adenylyl cyclase. Mutating either residue adjacent to Asp-1004 in human
DNA polymerase
(32) results in a phenotype remarkably similar to
that observed for the C441R and Y442H mutants. In both cases, the
mutant enzymes display low activity in the presence of
Mg2+, but normal activity in the presence of
Mn2+. In addition, the mutations in DNA polymerase
do
not alter the Km of the enzyme for the MgdNTPs,
indicating that the binding of nucleotides remains relatively
unaffected. Mutations of the catalytic aspartate residue (D1004A)
results in a non-functional DNA polymerase
, emphasizing the crucial
role of this residue in catalysis and paralleling the effects of this
mutation in adenylyl cyclase (31). These striking similarities are
likely the result of a shared catalytic mechanism between adenylyl
cyclase and DNA polymerase.
Our data on the metal dependence of the wild-type and mutant adenylyl
cyclases suggest a two-metal catalytic mechanism similar to that of DNA
polymerase (Fig. 4). According to this
model, the enzyme utilizes two Mg2+ ions, one associated
with the - and
-phosphates of ATP (denoted site B in Fig. 4) and
one catalytic Mg2+ ion primarily associated with the enzyme
(denoted site A). In analogy to DNA polymerase, we predict that the
Mg2+ at site A will coordinate to the 3'-hydroxyl of ATP,
promoting the nucleophilic attack of this group on the
-phosphate,
whereas the Mg2+ ion at site B assists in the leaving of
the pyrophosphate group and may also facilitate the cleavage of the
bond between the
- and
-phosphates by its electron-withdrawing
effect. Both metal ions are also responsible for stabilizing the
negative charge associated with the pentavalent transition state of
this chemical reaction. We propose that this model describes a highly
conserved catalytic mechanism for a diverse group of enzymes, including DNA and RNA polymerases as well as guanylyl and adenylyl cyclases, that
catalyze analogous phosphoryl transfer reactions.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. R. Green for the rabbit antibody specific for type V/VI adenylyl cyclase; J. Spanbauer for technical assistance; Dr. E. Fauman for help with molecular modeling analysis of the adenylyl cyclase structure and figures; and Drs. R. Neubig and M. Marletta for help with data analysis, thoughtful discussion, and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant GM53645 and by the Burroughs Wellcome Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Chemistry, 3301E MSRB III, University of Michigan Medical School, Ann
Arbor, MI 48109-0636. Tel.: 734-647-3634; Fax: 734-763-7799.
1
The abbreviations used are: G protein,
heterotrimeric guanine nucleotide-binding regulatory protein;
Gs, the
subunit of the G protein that stimulates
adenylyl cyclase; GTP
S, guanosine 5'-O-(3-thiotriphosphate); ATP
S, adenosine
5'-O-(1-thiotriphosphate).
2
1.2 × 105 yeast transformants
were obtained following transformation of yeast with a library of
randomly mutated adenylyl cyclases (library complexity = 4 × 105). 52 colonies were isolated for their ability to grow
in the presence of simultaneous forskolin and Gs
activation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|