Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.     

Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase

The structural organization of the low molecular mass form (43 kDa) of Bordetella pertussis adenylate cyclase was dissected taking advantage of the known sequence of the bacterial cya gene (Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbiol. 2, 19-30) and its low content of Trp and Met residues. Cleavage of the 43-kDa protein and of its complementary tryptic fragments (T25 and T18 peptides) with N-chlorosuccinimide and cyanogen bromide followed by sodium dodecyl sulfate-polyacrylamide gel analysis of digestion products allowed the following conclusions: (i) the catalytically active 43-kDa form of B. pertussis adenylate cyclase is within the first 400 residues of the protein encoded by the cya gene. T25 occupies the N-terminal domain of the protein (residues 1-235/237). Isolated T25 fragment exhibits a low but measurable enzymatic activity which indicates that it harbors the catalytic site; (ii) T18 which is the main calmodulin-binding domain, occupies the C-terminal segment of protein (residues 236/238-399) and is devoid of catalytic properties; (iii) the two complementary peptides T25 and T18 reassociated only in the presence of calmodulin, leading to significant recovery of the original activity. These results demonstrate that both fragments of the 43-kDa form of adenylate cyclase are essential for a high level of enzymatic activity.     

High-affinity calmodulin binding is required for the rapid entry of Bordetella pertussis adenylyl cyclase into neuroblastoma cells.

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains

The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a photoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:1 complex with CaM or azido-CaM. CaM-bound adenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically active conformation resembling that of the undigested complex. When proteolysis was carried out in the absence of CaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since this fragment cannot be cross-linked with azido-CaM we suggest that the CaM-binding site on the 25-kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.     

Site-directed mutagenesis of lysine 58 in a putative ATP-binding domain of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis abolishes catalytic activity

A 2.7-kb cya A gene fragment encoding the amino-terminal end of the calmodulin-sensitive adenylate cyclase from Bordetella pertussis has been placed under the control of the lac promoter for expression in Escherichia coli. Following induction with isopropyl beta-D-thiogalactoside, calmodulin-sensitive adenylate cyclase activity was detected in a cell extract from E. coli. The expression vector directed the synthesis of a 90-kDa polypeptide that was recognized by rabbit polyclonal antibodies raised against the catalytic subunit of B. pertussis adenylate cyclase. Inspection of the deduced amino acid sequence of the cya A gene product revealed a sequence with homology to consensus sequences for an ATP-binding domain found in many ATP-binding proteins. On the basis of the analysis of nucleotide binding proteins, a conserved lysine residue has been implicated in the binding of ATP. A putative ATP-binding domain in the B. pertussis adenylate cyclase possesses an analogous lysine residue at position 58. To test whether lysine 58 of the B. pertussis adenylate cyclase is a crucial residue for enzyme activity, it was replaced with methionine by oligonucleotide-directed mutagenesis. E. coli cells were transformed with the mutant cya A gene, and the expressed gene product was characterized. The mutant protein exhibited neither basal nor calmodulin-stimulated enzyme activity, indicating that lysine 58 plays a critical role in enzyme catalysis.     

Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy.

The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussis major exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC: tryptophan residues (W69 and W242), a nucleotide analogue (3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, Ant-dATP) and a cysteine-specific probe (acrylodan). CaM binding elicited large changes in the dynamics of W242, which dominates the fluorescence emission of both AC and AC-CaM, similar to that observed for isolated CaM-binding sequences of different lengths [Bouhss, A., Vincent, M., Munier, H., Gilles, A.M., Takahashi, M., Barzu, O., Danchin, A. & Gallay, J. (1996) Eur. J. Biochem.237, 619-628]. In contrast, Ant-dATP remains completely immobile and inaccessible to the solvent in both the AC and AC-CaM nucleotide-binding sites. As AC contains no cysteine residue, a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid, and only slightly affected by CaM. The protein's hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed significantly according to the probe used (19 ns for W242, 25 ns for Ant-dATP, and 35 ns for acrylodan), suggesting an elongated protein shape (axial ratio of approximately 1.9). These values increased greatly with the addition of CaM (39 ns for W242, 60-70 ns for Ant-dATP and 56 ns for acrylodan). This suggests that (a) the orientation of the probes is altered with respect to the protein axes and (b) the protein becomes more elongated with an axial ratio of approximately 2.4. For comparison, the hydrodynamic properties of the anthrax AC exotoxin were computed by a mathematical approach (hydropro), which uses the 3D structure [Drum, C.L., Yan, S.-Z., Bard, J., Shen, Y.-Q., Lu, D., Soelalman, S., Grabarek, Z., Bohm, A. & Tang, W.-J. (2002) Nature (London)415, 396-402]. A change in axial ratio is also observed on CaM binding, but in the reverse direction from that for AC: from 1.7 to 1.3. The mechanisms of activation of the two proteins by CaM may therefore be different.     

Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase

A truncated Bordetella pertussis cya gene product was expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose. Trypsin cleavage of the 432-residue recombinant protein (Mr = 46,659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties. The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19-kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin-dependent species. Reassociation was enhanced by calmodulin itself, which 'trapped' the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.     

Calmodulin-mediated adenylate cyclase from mammalian sperm

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.     

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.     

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.     

Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism

Calcium-saturated calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to a region in the C-terminal regulatory sequence of the enzyme to relieve autoinhibition. The structure of CaM in a high-affinity complex with a 25-residue peptide of CaMKI (residues 294-318) has been determined by X-ray crystallography at 1.7 A resolution. Upon complex formation, the CaMKI peptide adopts an alpha-helical conformation, while changes in the CaM domain linker enable both its N- and C-domains to wrap around the peptide helix. Target peptide residues Trp-303 (interacting with the CaM C-domain) and Met-316 (with the CaM N-domain) define the mode of binding as 1-14. In addition, two basic patches on the peptide form complementary charge interactions with CaM. The CaM-peptide affinity is approximately 1 pM, compared with 30 nM for the CaM-kinase complex, indicating that activation of autoinhibited CaMKI by CaM requires a costly energetic disruption of the interactions between the CaM-binding sequence and the rest of the enzyme. We present biochemical and structural evidence indicating the involvement of both CaM domains in the activation process: while the C-domain exhibits tight binding toward the regulatory sequence, the N-domain is necessary for activation. Our crystal structure also enables us to identify the full CaM-binding sequence. Residues Lys-296 and Phe-298 from the target peptide interact directly with CaM, demonstrating overlap between the autoinhibitory and CaM-binding sequences. Thus, the kinase activation mechanism involves the binding of CaM to residues associated with the inhibitory pseudosubstrate sequence.     

Sequential events in calmodulin on binding with calcium and interaction with target enzymes

khe conformational and functional events in calmodulin (CaM) are disproportionate to the mean saturation by Ca2+. The enhancement of intrinsic tyrosine fluorescence closely follows the appearance of species CaM X Can greater than or equal to 1; the exposure of the hydrophobic patch at the surface of CaM coincides with the appearance of CaM X Can greater than or equal to 2. For the activation of four different target enzymes, i.e., brain phosphodiesterase and adenylate cyclase, red blood cell Ca,Mg-ATPase, and skeletal muscle phosphorylase b kinase, CaM X Can greater than or equal to 3 is required. The different enzymes have the same affinity for the active species. The direct interaction of CaM with Ca2+ and phosphorylase b kinase has been analyzed according to the theory of energy coupling: whereas the first two stoichiometric calcium-binding constants in the complex are not significantly different from those of free CaM, the third Ca2+ binds with an affinity at least 10(6)-fold higher to enzyme-bound CaM than to free CaM, which corresponds to a free energy coupling of -7 kcal/mol CaM. The similarities in the activation mechanism of different enzymes suggest the existence of one unique CaM-binding domain. The characteristics of the interaction between CaM and melittin, a small amphiphatic cytotoxin, led us to propose melittin as a model for such a CaM-binding domain.     

Evidence for domain organization within the 61-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain.

The complete amino acid sequence of the 61-kDa calmodulin-dependent, cyclic nucleotide phosphodiesterase (CaM-PDE) from bovine brain has been determined. The native protein is a homodimer of N alpha-acetylated, 529-residue polypeptide chains, each of which has a calculated molecular weight of 60,755. The structural organization of this CaM-PDE has been investigated with use of limited proteolysis and synthetic peptide analogues. A site capable of interacting with CaM has been identified, and the position of the catalytic domain has been mapped. A fully active, CaM-independent fragment (Mr = 36,000), produced by limited tryptic cleavage in the absence of CaM, represents a functional catalytic domain. N-Terminal sequence and size indicate that this 36-kDa fragment is comprised of residues 136 to approximately 450 of the CaM-PDE. This catalytic domain encompasses a approximately 250 residue sequence that is conserved among PDE isozymes of diverse size, phylogeny, and function. CaM-PDE and its PDE homologues comprise a unique family of proteins, each having a catalytic domain that evolved from a common progenitor. A search of the sequence for potential CaM-binding sites revealed only one 15-residue segment with both a net positive charge and the ability to form an amphiphilic alpha-helix. Peptide analogues that include this amphiphilic segment were synthesized. Each was found to inhibit the CaM-dependent activation of the enzyme and to bind directly to CaM with high affinity in a calcium-dependent manner. This site is among the sequences cleaved from a 45-kDa chymotryptic fragment that has the complete catalytic domain but no longer binds CaM. These results indicate that residues located between position 23 and 41 of the native enzyme contribute significantly to the binding of CaM although the involvement of residues from additional sites is not excluded.     

Development of a novel photoreactive calmodulin derivative: cross-linking of purified adenylate cyclase from bovine brain

A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. 125I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca2+-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca2+-dependent stimulation of adenylate cyclase activity. 125I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca2+ dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with 125I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.     

Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Catalytic component of calmodulin-independent adenylate cyclase from bovine brain. Isolation and determination of partial amino acid sequence

The catalytic component of calmodulin-independent adenylate cyclase of cattle cerebral cortex was solubilized and purified to the homogeneous state. The conditions for preparative obtaining of the enzyme on the column with immobilized antibodies to adenylate cyclase were found. These antibodies were proved to interact with the calmodulin-independent rather than the calmodulin-dependent form of the enzyme. Molecular mass of the calmodulin-independent adenylate cyclase determined electrophoretically is 140 +/- 10 kDa. Amino acid composition of the enzyme and sequences of its fragments (in total 300 amino acid residues) obtained upon treatment with lysyl-specific proteinase from Achromobacter liticus were determined. Clone containing a cDNA 605 bp insertion coding for the 183 amino acid residue fragment of adenylate cyclase was isolated from the bovine brain cDNA library. Homology of this fragment to the known sequences of Escherichia coli and Bordetella pertussis adenylate cyclases was revealed.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.     

Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.     

Binding of 3'-anthraniloyl-2'-deoxy-ATP to calmodulin-activated adenylate cyclase from Bordetella pertussis and Bacillus anthracis

3'-Anthraniloyl-2'-deoxyadenosine 5'-triphosphate (Ant-dATP), a fluorescent analogue of ATP, was tested as a probe for the nucleotide-binding site of calmodulin (CaM)-activated adenylate cyclases from Bordetella pertussis (BPCYA47) and Bacillus anthracis (BACYA62). Ant-dATP competitively inhibited both bacterial enzymes expressed in Escherichia coli (ki approximately 10 microM). Binding of the analogue to adenylate cyclase was monitored by equilibrium dialysis and by an increase in its fluorescence emission at 420 nm upon excitation at 330 nm. Whereas the fluorescence of Ant-dATP was little influenced by divalent cations, CaM, or adenylate cyclase alone, the Ca2+.CaM.cyclase complex increased up to 4 times the quantum yield of Ant-dATP. Binding of the analogue to the catalytic site of BPCYA47 and BACYA62 was specific as shown by its displacement with ATP or 3'-dATP. Our results substantiate the role of CaM in favoring substrate binding to CaM-activated enzymes.