Bordetella pertussis Adenylate Cyclase - The Journal of Biological ...

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Dec 26, 1991 - Of the 9 histidines located in the catalytic domain of. Bordetella pertussis adenylate cyclase, three (Hise3,. Hisloe, and Hiszs8) were found to be ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 267, No.14, Issue of May’ 15,, pp. 9816-9820, 1992 Printed in U.S.A.

0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

The Roleof Histidine 63 in the Catalytic Mechanism of Bordetella pertussis Adenylate Cyclase* (Received for publication, December 26,1991)

Helene Muniert, Ahmed Bouhsst, Evelyne KrinQ, Antoine DanchinQll,Anne-Marie Gillest, Philippe GlaserQ, and Octavian BPrzuSlI From the $Unite de Bwchimie des Regulations Cellulaires, §Unite de Regulation de I’Expresswn Genktique, Institut Pasteur, 75724 Paris Cedex 15, France

Of the 9 histidines located in the catalytic domain of Bordetella pertussis adenylate cyclase, three (Hise3, Hisloe, and Hiszs8)were found to be conserved in the adenylate cyclase of Bacillus anthracis, another calmodulin-dependent enzyme. Substitution of Hise3with Arg, Glu, Gln, or Val decreased the catalytic efficiency of adenylate cyclase between 2 and 3 orders of magnitude and altered the kinetic properties of the enzyme. These effects varied in relation to the nature of the substituting residue, pH, and direction of the reaction, Le. ATP cyclization (forward) or ATP synthesis (reverse). Arg was the best substituent for Hises as catalyst in the forward reaction, with shiftof the optimum pH to the alkalineside,whereas Glu was the best substituent for Hise3 in the reverse reaction, with shift of the optimum pH to the acidic side. Diethyl pyrocarbonate, which had a deleterious effect on wild-type adenylate cyclase was ineffective on Hise3 mutants. From these results we conclude that Hise3is involved in the reaction mechanism of adenylate cyclase, which requires a general acidbase catalyst, most probablyas an intermediate in a charge-relay system.

Adenylate cyclase occupiesa singular place among the large and heterogeneous family of ATP-utilizing enzymes in that the formation of the new chemical bond is an intramolecular event. Stereochemical studies using phosphorothionate analogs of ATP or GTP showed that the reaction catalyzed by bacterial or mammalian adenylate/guanylate cyclases proceeds with inversion of the configuration at thea-phosphorus (1-4). Gerlt et al. (1) proposed that cyclization occurs by a single nucleophilic displacement and that a general base catalyst is required to assist in the ionization of the 3”OH group of the pentose moiety, which is supposed to attack the aphosphorus atom of nucleoside triphosphate. A candidate for such a role is the imidazole side chain of the histidine residue, due to itsability to subtract protonsat pH values close to the optimum pH of the reaction catalyzed by these enzymes. Adenylate cyclase of Bordetella pertussis is a calmodu*This work was supported by Centre National de la Recherche Scientifique Grant URA Dl129 and Institut National de la Sant6 et de la Recherche Medicale Grant INSERM CRE 910615. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. 1To whom correspondence should be addressed Unit6 de Biochimie des Rigulations Cellulaires, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-45-68-84-05;Fax: 33-1-43-06-98-35.

lin(CaM)’-activated enzyme whosecatalytic domain is located within the first 400 amino acid residues (5,6). From the 9 His residues occupying the positions 5, 46, 63, 106, 108, 141, 197, 288, and 298, 3 (His63,Hido6, and Hiszg8)were found to be conserved in the adenylate cyclase of Bacillus anthracis, another CaM-activated enzyme (7). Replacement of Hiszg8with Leu or Arg dramatically decreased the activity of adenylate cyclase from B. pertussis. However, the kinetic characteristics of modified adenylate cyclase did not support the idea that Hiszg8might be involved directly in the catalytic step (8). In this paper, we analyzed by site-directed mutagenesis the role of the other two conserved His residues of B. pertussis adenylate cyclase. We show that His63,albeit not essential for the binding of nucleotide, plays an important role in the cyclization process of ATP, as suggested by a profound alteration of kinetic properties of enzyme that has been modified at this residue. EXPERIMENTALPROCEDURES

Chemicals-Adenine nucleotides, substrates, restriction enzymes, and T4 DNA ligase were obtained from Boehringer Mannheim. T7 DNA polymerase and the four deoxyribonucleoside triphosphates used in sequencing reactions were from Pharmacia. CaM-agarose, bovine brain CaM, dansyl-CaM, and diethyl pyrocarbonate were from Sigma. Urea (fluorimetrically pure) was a product of Schwartz Mann. [w3’P]ATP (3,000 Ci/mmol), [w3’P]-dATP (1,000 Ci/mmol), ATP (1,000 Ci/mmol), and [3H]cAMP (40 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, United Kingdom. Bacterial Strains and GrowthConditions-The Escherichia coli strainTG1(9) was used for sequence analysis and site-directed mutagenesis. Production of recombinant protein was performed using the protease-deficient strain Y1083 BNN103 (lo), which harbors the plasmid pDIA5227, or a derivative harboring the plasmid pDIA17. Plasmid pDIA5227 encodes a 432-residue truncated adenylate cyclase of B. pertussis (11).Plasmid pDIA17 expresses the laclgene inserted in the tetracycline gene of plasmid pACYC184 such that itis, at least in part, under the control of the tetracycline promoter. Cultures were grown in LB medium (12) supplemented with 100 pg/ml ampicillin and 20 pg/ml chloramphenicol. Synthesis of truncated adenylate cyclase was induced by isopropyl-P-D-thiogalactoside(1 mM) when cultures reached an optical density of 0.5. Bacteria were harvested by centrifugation 4 h after induction. Site-directed Mutagenesis and SequenceAnalysis-Oligonucleotide-directed mutagenesis was performed on the single-stranded form of pDIA5227 using the Amersham system based on the method of Taylor et al. (13). The histidine codon at position 63 (CAC) was modified tothe glutamine (CAA), arginine (CGT), glutamic acid (GAA),and valine (GTA) codons using the following oligonucleotides.

9816

a

5’TTG GGC GTG GCC AAG 5’TTG GGC GTG GCC AAG 5’TTG GGC GTG GCC AAG 5’TTG GGC GTG GTA GCC AAG The abbreviation used is: CaM, calmodulin.

TCG3’, TCG3’, TCG3’, TCG3’

Adenylate B. pertussis The histidine codon at position 106(CAC)wasmodified tothe glutamine (CAA), asparagine (AAC), and arginine (CGA) codons using the following oligonucleotides.

&

5'AGC CTG GCG GGC CATB', 5'AGC CTG GCG A E GGC CAT3', 5'AGC CTG GCG CGA GGC CAT3'

Cyclase

9817

TABLE I Catalytic activity of His-modified forms of B. pertussis adenylate cyclase determined in the forward direction at two pH values Wild-type and His-modified enzymes were purified by CaM-agarose chromatography. Activity Enzyme

pH 8 pH 10 For each mutagenesis experiment, the whole sequence of the cya gene was screened for the absence of any other mutations by the dideoxypmollmin. mg ofprotein" nucleotide sequencing method (14). WT 1980 (100) 820 (100) Purification and Assay of Adenylate Cyclase-Truncated B. pertusH63R (15.8) 15 130 (0.76) sis adenylate cyclase expressed in E. coli was purified by affinity H63Q 8.3 (0.42) 90 (10.9) chromatography on CaM-agarose, essentially as described by Glaser H63V 13.0 (0.66) 47 (5.7) et al. (15). Enzyme activity in the forward direction (ATP cyclization) 18 (2.2) H63E(0.52) 10.3 was measured using the procedure of White (16), as described previously (6). Reverse adenylate cyclase activity was measured by couH106R (34.1) 675 151 (18.4) pling the synthesis of ATP from cAMP and PPI to the reaction 205 (10.4) 80 Hl06Q (9.8) catalyzed by hexokinase and glucose-6-phosphate dehydrogenase and (8.2) 24867 (12.5) H106N measuring the increase in absorption due to theformation of NADPH a Activity was determined at 30 "C, 5 mM ATP and 1 p~ CaM at 334 nm. The reaction medium (final volume of 0.5 ml) contained 50 mM buffer (Tris maleate for pH 6; Tris-HC1 for pH 7.4 and 9), 6 concentrations in Tris-HC1 buffer (pH 8) or glycine/NaOH buffer mM MgC12, 0.4 mM NADP+, 1 mM glucose, variable concentrations (pH 10). The numbers in parentheses are relative values, the cyclase of cAMP and sodium pyrophosphate, 1 p M CaM, and 2.5 units of activity of the wild-type protein being considered as 100%. each hexokinase and glucose-6-phosphate dehydrogenase. The reaction was triggered by the addition of adenylate cyclase (2-5 p1 of enzyme solution) diluted with buffer a t the desired pH value, to which 0.1% Triton X-100 was added. One unit of adenylate cyclase activity corresponds to 1 pmol of cAMP formed or consumed in 1 min at 30 "C. Modification of Adenylate Cyclase with Diethyl PyrocarbonateAdenylate cyclase (20 p~ in 0.1 M phosphate buffer, pH 6.0)was treated with diethyl pyrocarbonate diluted with anhydrous acetonitrile (17). Acetonitrile (final concentrations not higher than 2%) did not affect enzyme activity. Incubation was carried out at 4 "C, and at different time intervals, aliquots of the reaction mixture were withdrawn for assay of enzyme activity in the reverse direction. Analytical Procedures-Protein concentration was determined according to Bradford (18)using a Bio-Rad kit. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was performed as described by 50-10 Laemmli (19). Fluorescence measurements were done with a PerkinElmer L S d B luminescence spectrometer thermostated at 25 "C using 1 X 1-cm UV-grade quartz cuvettes(sample volume of 2 ml). Emission spectra of adenylate cyclase (Aexc. = 295 nm) were recorded from 305 to 400 nm.

RESULTS

From the 2 His residues targeted in this work as being importantin expressing efficient catalysis in B. pertussis adenylate cyclase, one proved to be critical. The adenylate cyclase activity of His63 mutants determined at pH 8, and nearly saturating concentrations of ATP and CaM, showed between 2 and 3 orders of magnitude decrease in catalytic activity (Table I). All modified forms of adenylate cyclase exhibited fluorescent properties similar to those of the parent protein (11)and identical sensitivity toward denaturation by urea (data not shown). On the other hand, the highest Kd value for CaM of these modified forms was 0.7 nM, in contrast to the Kd for CaM of the wild-type enzyme, which is 0.2 nM. It is possible that the former value, determined from the activation curve of enzymes by CaM, is an overestimate. Enzyme concentration in the assay mixture was high in the case of the H63 mutants, to ensure accurate determinations of activity. The residual activity of Hi~~~-modified forms of adenylate cyclase measured at pH 8 differed little on the substituting residue. However, at more alkaline pH values, the relative activity of HiP-modified forms of adenylate cyclase as compared with the wild-type protein was considerably higher than at pH 8 and was dependent on the substituting residue. This strong "pH effect" was unique to H63 mutants. In the case of His'OG-modifiedforms of adenylate cyclase the relative activity as compared to wild-type protein was roughly similar at both

I

-1.0

-0.5

0

0.6

1.0

1.5

2.0

2.5

FIG. 1. Double-reciprocal plots of wild-type (W) and H63 variant (0)of B. pertussis adenylate cyclase in the forward direction andat two different pH values. The broken line in the upper part is a curve calculated from the V , and KiTpvalues of the H63R mutant determined by fitting the experimental data into the following rate equation: u = V,. [ATP]/(K$Tp + [ATP] [ATPIZ/ K,). Numbers on the right side of the abscissa correspond to thewildtype enzyme, whereas numbers on the left side of the abscissa correspond to the H63R mutant.

+

pH 8 and 10. Determination of the reaction rates as afunction of ATP concentration showed that at pH 8 the wild-type adenylate cyclase exhibited a Michaelian dependence, whereas the H63R mutant was inhibited by excess ATP (Fig. 1).The reaction rate of the latter form of adenylate cyclase, as a function of ATP concentration, obeyed the following equation, v = V"

[ATPI [ATP] + Kim + [ATP]'/KI

which allowed the calculation from experimental data of the V,,, (85 pmol/min-mg of protein), KkTp (0.6 mM) and Kr (1.2 mM). At pH 10, all adenylate cyclase variants obeyed Mi-

9818

Adenylate B. pertussis

chealis-Menten kinetics. The absence of inhibition by excess ATP might be due to a 10- to 20-fold increase in apparent K , for nucleotide substrates (Fig. 1). These results showed the following: (i) His63 apparently does not participatein the binding of the nucleotide substrate, since the K , for ATP was not affected by substitution of this residue with Arg (as well as with Gln, Glu, or Val); (ii) a charged residue deprotonated around pH 10 is important for the binding of ATP, since the K, for nucleotide was increased by more than an order of magnitude at this pH, for both the wild-type and theHis-modified enzymes. Inhibition of His63mutants by excess ATP at pH 8 might reflect an intrinsicproperty of the enzyme that becomes apparent when this aromatic charged residue is replaced by other residues. This prompted us to examine the reaction rates at several pH values, in thedirection of both the disappearance of and the formation of ATP. The pH dependence of the wild-type adenylate cyclase activity in the forward direction (CAMP formation) is shown in Fig. 2. The K, for ATP increased gradually from pH 6 to 8 and then increased more rapidly from pH 8 to 10; the kc, reached its highest value at pH9. Similar kinetic behaviors, as a function of pH, were shown by the His'OG mutants (data not shown). Under identical experimental conditions, the H63R mutants exhibited K , values for ATP close to thatof the wild-type protein, but the V , values were shifted to the alkaline pH side as compared with the wild-type enzyme (Fig. 2). It should be noted that at pH values lower than 7, the wild-type adenylate cyclase wasalso inhibited by ATP concentrations higher than 10 mM. These results showed that His63is indeed involved in the catalysis of adenylate cyclase. This conclusion was strengthened by the analysis of the reaction in the direction of ATP formation (Fig. 3). At fixed concentrations of cAMP and inorganic pyrophosphate, the adenylate cyclase activity was maximal at pH 7.5, irrespective of thenature or site of mutation. A single exception was noted, in that the activity of the H63E mutant was maximal at pH 6. The ratio of the reaction rate at pH 9 to that at 6pH was significantly different for wild-type enzyme (0.03), H63R mutant (3.03), H63Q mutant (2.34), H63V mutant (2.43), and H63E mutant (0.068). A more detailed kinetic analysis of the reaction catalyzed by the wild-type and Hi~~~-modified forms of adenylate cyclase at pH 6 andin the direction of ATP formation again revealed

Cyclase

PH

.,

FIG.3. Dependence on pH of the reverse reaction catalyzed by B. pertussis adenylate cyclase (pmol/min*mgof protein) at a fixed concentration of cAMP (10 mM) and pyrophosphate (2 mM). 0, wild-type enzyme; H63E mutant; 0, H63R mutant; 0, H63V mutant.

TABLE I1 Kinetic parameters of wild-type and H63 mutantsof B. pertussis adenylate cyclase in the reverse reaction at pH6 katwas calculated assuming a molecular mass of 46,659 daltons. The numbers in parentheses are relative values, the cyclase activity of the wild-type protein being considered as 100%. Enzyme

S-'

WT H63E H63R H63V H639

120 10.3 1.04 0.63 0.52

kat

Zpi

L,

PP

k, ZmPi

m Mm M

2.0 3.9 2.5 3.5 2.8

3.4 14.3 15.4 19.6 16.3

61.5 (100) 2.64 (4.3) 0.42 (0.7) 0.18 (0.3) 0.19 (0.3)

35.3 (100) 0.72 (2.0) 0.067 (0.2) 0.032 (0.09) 0.032 (0.09)

interesting differences. The kcat of the H63E mutant represented 8.5%of that of the wild-type enzyme, whereas all other Hi~~~-modified forms of adenylate cyclase exhibited katvalues less than 1%of that of the parentprotein. On the other hand, the K , for PPIwas significantly higher for all His63mutants as compared with the wild-type enzyme, whereas the K, for cAMP varied by a factor of only 2 or less (Table 11). Having inferred the catalytic role of His63by site-directed mutagenesis, we examined the effect of diethyl pyrocarbonate on the activity of the wild-type, H63Q and H106R mutants of adenylate cyclase (Fig.4). Diethyl pyrocarbonate exerted a deleterious effect on both the wild-type and the H106R mutant, being practically ineffective on the H63Q mutant. Although we did notundertakea more detailed analysis of adenylate cyclase inhibition by diethyl pyrocarbonate, we suppose that modification of the single His63residue is responsible for the inactivation of wild-type adenylate cyclase by this reagent. DISCUSSION

Histidine participates in a large number of enzymatic reactions by a variety of mechanisms, which include general acidbase catalysis, electrophilic catalysis, and binding of the FIG.2. Dependence on pH of kat(circles) and of KiTP substrate via hydrogen bonding and/or electrostatic interac(squares) of wild-type (opensymbols) and of the H63R mutant tions (20-25). As different amino acid residues canexert (closed symbols) of 33. pertussis adenylate cyclase. katwas calculated assuming a molecular mass of 46,659 daltons. Numbers on several, but not all, of these functions, we replaced His63,and the left side of the abscissa (representing kat)correspond to the wild- His'" in B. pertussis adenylate cyclase with either Gln/Asn, type enzyme, those onthe right side correspond to theH63R mutant. Glu, Arg, or Val. The first 2 amino acids lack proton transfer

B. pertussis Adenylate Cyclase

9819

being the catalytic triad Asp-His-Ser found in serine-proteases. A direct participation of His63in the reaction catalyzed by B. pertussis adenylate cyclase is favored by the differential effect of Arg and Glu substitutions on the forward and reverse reactions at alkaline and acid pH values. Thus, Arg, the best substituent for His63 ascatalyst in the forward reaction, shifted the optimum pH of B. pertussis adenylate cyclase reaction to a more alkaline value. On the other hand, Glu, the best substituent for His63in the reverse reaction, shifted the optimum pH to the acid side. In this respect, the optimum pH of the reaction catalyzed by B. pertussis was successfully "tailored." Several arguments might be considered for an indirect participation of His63in a general acidbase catalytic mechanisms: (i) replacement of His63by Gln/Asn or Val did not generate inactive (or almost inactive) species of enzyme, as it was expected. At pH 8, the activity of the H63Q and IO 20 30 H63V mutants in the forward direction was not very different T I M E , 11111. FIG. 4. Effect of diethyl pyrocarbonate (0.4 mM) on the from that of the H63E or H63R mutants. (ii) The kinetic activity of wild-type (O), H106R (A),and H63Q (W) forms of characteristics of all four His63 mutants, i.e. inhibition by excess of ATP, were rather similar from both qualitative and B. pertussis adenylate cyclase. quantitative points of view. (iii) The marked increase in the capability of His, but are more or less sterically conservative hydroxide dependence of ATP cyclization between pH 8 and substituents with similar polar effects. Glu and Arg, charged 10, compared with the wild-type enzyme, favor the idea that amino acid residues, are capable of participating in general another general acidbase catalyst that has apK, higher than 9 might be involved in the attack of the nucleotide substrate. acidbase mechanisms, hydrogen bonding, orelectrostatic Tyrosine seems to be such a candidate for several reasons. A t interactions, whereas Val lacks all of these capabilities. The replacement ofHis"byArg, Gln, or Asn decreased the pH 9, which is the maximum for B. pertussis adenylate cyclase catalytic efficiency of adenylate cyclase by between 65 and activity, this residue is in the protonated form and is therefore 90%. The other kinetic properties of the His" mutants were ineffective in cyclization of ATP, in the absence of a chargeted of Tyr at pH9 or similar to that of the wild-type protein, which seems to relay system. H i ~ ~ ~ - a s s i sdeprotonation below would permit an efficient subtraction of the proton exclude this residue as playing an essential role in the cyclization of ATP. It is difficult to say, based on fluorescence from the 3'-OH group in the ATP molecule and promote measurements, if altered activity of these mutants are second- subsequent cyclization. In the absence of His63and, independary or not to conformational effects on the protein upon ent of its substituting residue, Tyr deprotonation will occur amino acid substitutions. Only high resolution data (e.g. x- only at alkaline pH values, thus explaining the marked increase in the hydroxide dependence of catalysis in the forward ray analysis) are able to eliminate such uncertainties. The most significant effects on the kinetic properties of B. direction between pH 8 and 10, as compared with the wildpertussis adenylate cyclase were observed upon substitution type enzyme. Another observation emerging from our experiments on of His63.These effects varied upon the nature of the substituting residue, pH, and direction of the reaction, i.e. ATP wild-type and His63mutants of B. pertussis adenylate cyclase synthesis or cyclization, and were directly correlated with the is the inhibition by excess ATP. Although this phenomenon sensitivity of adenylate cyclase to inhibition by diethyl pyro- was not yet observed with B. pertussis enzyme, it has been carbonate. To rationalize the observed effects of the His63 found previouslywhen adenylate cyclasefrom E. coli was mutation on the activity of B. pertussis adenylate cyclase, examined (26). There are several mechanistic explanations several points should be remembered (i)the equilibrium for excess ATP inhibition of adenylate cyclase. One explanation is the presence of two ATP-binding sites on the bacconstant (Kq = [cAMP][PP,][H']/[ATP]) of the reaction at 30 "cand 10 mM MgCl' (2 X lo-' M')' favors the formation terial enzyme, one with a high affinity for nucleotide (the of cAMP at all pH values between 6 and 10. At physiological catalytic site), the second with a lower affinity for nucleotide pH (7.4) and 3 mM ATP (which is also physiologically en- (the regulatory site). However, binding studies by equilibrium countered in living cells), the conversion of nucleotide to dialysis using noncyclizable ATP analogs (3"dATP or 3'cAMP and PPi is 94.7%. At the two extreme pH values (6 anthraniloyl-dATP) on both B. pertussis and B. unthrucis and 10) used in our experiments, this conversion would be 55 adenylate cyclase identified a single nucleotide-binding site and 99.85%, respectively. (ii) Assuming that a general acid/ on these enzymes (27, 28). Another explanation would be the base catalyst is involved in the interconversion of ATP and formation of an abortive complex, favored by the substitution CAMP, His is the best target for such a function due to its of His63 with other amino acid residues, thereby causing ability to subtract (forward reaction) or release (reverse re- substrate inhibition. action) protons at pHvalues close to the optimum pH of the In conclusion, from the three His residues conserved in B. reaction catalyzed by adenylate cyclase. (iii) His can exertits pertussis and B. unthrucis adenylate cyclase, twoare essential catalytic function by either interacting directly with the sub- for expressing full catalytic activity. One of them (Hiszg8)is strate or being an intermediate in a two- or three-member most probably involved in the mechanism of activation of proton shuttle system. Such a charge-relay catalytic system bacterial enzyme by CaM (8).His63is directly involved in the was frequently seen in the reactions catalyzed by hydrolases reaction mechanism, most probably as part of a charge relay or transferases (20-25), the most illustrative example in this catalytic system. Tasks for future research will be to identify and clarify the role of other amino acid residues playing a role 0.Btrzu, unpublished data. in such a proton shuttle system.

9820

B. pertussis Adenylate Cyclase

Acknowledgments-We thank Marcia Goldberg for carefully reading this manuscript and Mireille Ferrand for excellent secretarial help.

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