Sep 9, 1992 - Leonard, N. J. (1984) Biochemistry 23, 4350-4357. 10. Buecher, J. A., Vedvick ... Gibbs, C. S. & Zoller, M. J. (1991) J. Biol. Chem. 266, 8923-.
Proc. Nati. Acad. Sci. USA Vol. 90, pp. 442-446, January 1993 Biochemistry
The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP (enzyme mechanism/protein-tyroslne kinase/phosphorylation)
ANA C. CARRERA, KIRILL ALEXANDROV, AND THOMAS M. ROBERTS* Dana-Farber Cancer Institute, Department of Cellular and Molecular Biology, Harvard Medical School, Boston, MA 02115
Communicated by Ruth Sager, October 21, 1992 (received for review September 9, 1992)
ABSTRACT The study of the various protein kinases reveals that, despite their considerably diversity, they have evolved from a common origin. Eleven conserved subdomains have been described that encompass the catalytic core of these enzymes. One of these conserved regions, subdomain II, contains an invariant lysine residue present in all known protein kinase catalytic domains. Two facts have suggested that this conserved lysine of subdomain II is essential for binding ATP: (i) several investigators have demonstrated that this residue is physically proximal to the ATP molecule, and (ig) conservative substitutions at this site render the kinase inactive. However, these results are also consistent with a functional role of the conserved lysine of subdomain H in orienting or facilitating the transfer of phosphate. To study in more detail the role of subdomain II, we have generated mutants of the proteintyrosine kinase pp56k4k that have single amino acid substitutions within the area surrounding the conserved residue Lys273 in subdomain II. When compared with wild-type pp56kk, these mutants displayed profound reductions in their phosphotransfer efficiencies and small differences in their affinities for ATP. Further, the substitution of argnine for Lys-273 resulted in a mutant protein unable to transfer the -phosphate of ATP but able to bind 8-azido-ATP with an efficiency similar to that of wild-type ppS6"k. These results suggest that the region including Lys-273 of subdomain II is involved in the enzymatic process of phosphate transfer, rather than in anchoring ATP.
observed in several PKs upon site-directed mutagenesis of this conserved lysine of subdomain 11 (11, 12), together with the previous data, supported the idea that the conserved lysine of subdomain II is essential for the binding of ATP. However, as other investigators have pointed out (11), all the evidence presented to date is also consistent with the possible participation of this subdomain in the actual mechanism of phosphate transfer. A second area of the kinase domain that has been implicated in ATP binding is the glycine-rich loop of subdomain I (10). This loop, displaying the consensus sequence Gly-XaaGly-Xaa-Xaa-Gly, is close to the phosphates of MgATP in the crystal structure of the cAMP-dependent kinase (6). The nearly invariant Gly-50 and Gly-52 fall within this consensus sequence. This motif is part of the Rossmann fold structure associated with many nucleotide binding sites (13). A similar motif containing a glycine-rich loop is found in proteins as diverse as adenylate kinase (14), GTP-binding proteins such as p2lms (P loop; ref. 15), hexokinase (16), HSC70 (17), and actin (18). The single motif common to all these nucleotidebinding proteins is the glycine-rich motif, suggesting that it may serve as phosphate anchor (19). The available data on the functional role of subdomains I and II of the catalytic core of PKs do not clearly establish whether the conserved lysine of subdomain II is essential for ATP binding or whether its major role is related to the actual transfer of phosphate. To analyze the role of subdomain II, we have studied 10 mutants with single amino acid substitutions in the vicinity of Lys-273 of the lymphoid proteintyrosine kinase pp56lck.
Protein kinases (PKs) are phosphotransferases that catalyze the transfer of the y-phosphate of ATP to an amino acid side chain (for review see refs. 1-7). Sequence similarities define two major units in the family of PKs: a conserved catalytic core and nonconserved flanking regions (1). The peripheral nonconserved regions flanking the catalytic core are important for functions such as regulation and subcellular localization (1, 2). The sequence alignment of the catalytic domains of PKs (1) reveals that the conservation is not uniform but, rather, consists of alternating regions of high and low homology. Eleven major conserved subdomains have been identified (I to XI), which are separated by regions of lower conservation (1). The first clue for localizing the ATP binding site within the catalytic core came from studies on the cAMP-dependent kinase. Affinity labeling with the ATP analog 5'-(pfluorosulfonylbenzoyl)adenosine (FSBA; ref. 8) inhibited the enzyme by covalently modifying Lys-72 (8), a conserved residue of subdomain II. FSBA contains a reactive group at a position that approximates the y-phosphate of ATP. The proximity of Lys-72 to the y-phosphate of ATP was confirmed by other investigators (6, 7, 9, 10). The inactivation
EXPERIMENTAL PROCEDURES Mutant Construction. To prepare substitution mutants in subdomain II of pp56lck, we used syn-k, synthetic Ick gene encoding pp56lck (unpublished work), cloned into Gex-2T vector (20). Mutants have been generated by replacement of the codons encoding Lys-269 to Lys-276 by the corresponding synthetic fragment containing an 8% level of sequence degeneracy. Escherichia coli JM109 (Stratagene) colonies transformed with the mixture of mutated constructs were picked and screened for overexpression of full-length molecules. DNA preparations obtained from the bacterial colonies producing full-length proteins were then sequenced. Ten mutants, corresponding to single amino acid substitutions between Lys-269 and Lys-276, were selected for further analysis. Analysis of Protein Production. Several parameters were optimized for induction of protein production to achieve maximal activity and solubility of the protein. Soluble and
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Abbreviations: FSBA, 5'-(p-fluorosulfonylbenzoyl)adenosine; GST, glutathione S-transferase; PK, protein kinase. *To whom reprint requests should be addressed.
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Biochemistry: Carrera et al. insoluble fractions were separated by centrifugation at 13,000 x g for 30 min at 40C. The amount of pp56lck protein present in each fraction was estimated by Western blotting (as in ref. 21) using anti-pp561ck antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The activity of the soluble products was analyzed in kinase reactions using enolase as a substrate (as below). The optimal conditions for enzyme solubility and activity were obtained with overnight cultures of E. coli X90 (22) diluted 1:10 and incubated for 4 hr in TY medium at 370C (in the absence of isopropyl 8-D-thiogalactopyranoside). Cells were recovered by centrifugation and suspended in 1% (vol/vol) Triton X-100 with phenyl methylsulfonyl fluoride (2 mM), aprotinin (0.15 unit/ml), leupeptin (2.5 mg/ml), pepstatin (10 pg/ml), NaF (5 AM), and Na3VO4 (2 mM) at 40C. Sonication was performed by two rounds of 10-sec pulses (5 min apart) at a duty cycle of 70% and output control of 5 (Heat Systems Ultrasonics, Farmingdale, NY, model W225). The glutathione S-transferase (GST)-pp56lck fusion protein (G-pp56lck) was purified as described (20). Protein concentration was estimated by Coomassie brilliant blue R(Sigma) staining of SDS/polyacrylamide gels, with bovine serum albumin as standard, or by binchoninic acid (BCA) assay (Pierce). Western blot analysis was performed as reported (21). Anti-phosphotyrosine antibodies were prepared by B. Druker in our laboratory. Kinase Reactions, Data Analysis, and Photoaffinity Labeling. For kinase reactions, 10 ,l containing 50 ng of purified kinase was preincubated at 25°C for 1 min and mixed with 20 ,ul of 2x kinase reaction cocktail and 10 ,ul of acid-denatured enolase (at the appropriate concentration). The cocktail contained 50 mM Tris-HC1 (pH 7.4), 10 mM MnC12, and the appropriate dilution of the ATP stock (100 ,uM ATP, 10 ,Ci of [y-32P]ATP per ,ul, 3000 Ci/mmol, NEN/DuPont; 1 Ci = 37 GBq). Reaction mixtures were incubated at 25°C for 2 min (mixed every 30 sec), and reactions were terminated by addition of 10 ,ul of 100 mM EDTA (pH 8.0). For the comparison of G-pp56Ick and baculoviral pp56lck (30%o pure; ref. 23), reaction mixtures were incubated for 5 min. Substrate and enzyme were resolved by SDS/PAGE. For the determination of kinetic parameters, phosphate incorporated into enolase was quantitated by liquid scintillation counting. Vmx and Km were estimated by graphic methods (24, 25). Photoaffinity labeling of G-pp56Ick was performed as described (26).
RESULTS Mutant Construction. To distinguish between a passive role of subdomain II in anchoring ATP or a catalytic role in the phosphorylation reaction, we chose to analyze the kinetic consequences of introducing single amino acid substitutions (conservative or nonconservative) at residues located between Lys-269 and Lys-276 of pp56Ick. Wild-type and mutated genes encoding pp56Ick were cloned in p-Gex-2T to facilitate production of mutant proteins referred to as G-pp561ck in bacteria and subsequent purification. Fig. 1A illustrates the area of subdomain II of pp56lck examined, as well as the various single amino acid substitutions selected for the study. G-ppS6I-k Protein Production, Purification, and Enzymatic
Characterization. To compare mutants in subdomain II with wild-type G-pp56lck, we first optimized the expression of the wild-type enzyme in bacteria (see Experimental Procedures). The optimal conditions yielded maximal activity (see below for comparison with baculoviral pp56Ick) and solubility (=90%). Using these conditions, we compared the lysates of nontransformed E. coli X90 (C3) with lysates of X90 bacteria expressing wild-type fusion protein G-pp561ck (WT), mutated G-pp561ck (see nomenclature in Fig. lA), a GST-Ser/Thr kinase Raf-1 fusion protein (Cl), and GST (C2). Analysis of
443
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FIG. 1. Comparison of E. coli lysates containing wild-type or mutated G-ppS6lck: (A) Representation of the positions of wild-type pp5ock that have been substituted (upper line) and the corresponding amino acids introduced (lower line). Numbers in parentheses represent the positions of the residues relative to Lys-273. Substitutions of Lys-273 were named Ml (Lys-273 -. Arg), M2 (Lys-273 -. Asn), and M3 (Lys-273 -+ Met). (B-D) E. coli X90 bacteria were transformed with constructs encoding wild-type (WT) or singlesubstitution mutants of G-pp56ock (named as in A), GST-Ser/Thr kinase Raf-1 fusion protein (Cl), or GST (C2) or with medium alone (C3). Bacteria were induced and lysed, and soluble proteins were recovered by centrifugation. Fifteen microliters of each lysate was loaded on an SDS/10%o polyacrylamide gel and analyzed by Western blotting using anti-pp561ck antibodies (B), Coomassie blue staining (C), or Western blotting using anti-phosphotyrosine antibodies (D). MW, molecular weight markers (Mr x 10-3 at left).
15 pul of each lysate by Western blotting with anti-pp56Ick antibodies revealed that bacteria transformed with the various G-pp561ck constructs, but not the controls, contained similar amounts of G-pp561ck (Fig. 1B, an 82-kDa band corresponding to 26 kDa of GST fused to 56 kDa of pps6lck). Further, a similar protein composition was found (Fig. 1C) when the same volume of the various preparations of X90 bacterial lysates (containing the various constructs) were analyzed by Coomassie blue staining. In contrast, different intensities of phosphotyrosine signal were detected when similar volumes of the lysates were compared by antiphosphotyrosine Western blotting (Fig. 1D). These results indicate that the different mutants display different kinase activities. G-pp56Ick was purified as described (20). This procedure yielded apparently pure wild-type or mutated G-pp561ck as judged by Coomassie blue staining (Fig. 2A). Purified wildtype G-pp56lck was highly active as estimated by autophosphorylation (Fig. 2B). The Gex-2T-syn-k construct encoding G-pp561ck includes in its sequence a protease site (thrombin)
Biochemistry: Carrera et al.
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Proc. Natl. Acad. Sci. USA 90 (1993)
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FIG. 2. SDS/PAGE analysis of purity and autokinase activity of purified G-pp,6Ick: Wild-type (WT) and Lys-273 Arg mutant (K273R) G-pp56Ick proteins were produced in E. coli X90. The fusion protein present in the bacterial lysates was purified by using glutathione-Sepharose beads (200 ng of pure protein obtained from 400 /kg of the total soluble protein fraction). Purified WT and K273R G-pp561ck were analyzed by SDS/PAGE followed by Coomassie blue staining (A). Samples were also tested for their autophosphorylating activity in vitro and resolved by SDS/PAGE. The resulting gel was analyzed by autoradiography (B). MWM, molecular weight markers (Mr 10-3 at right in A). -.
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located between the GST fragment and pp5sck. We also compared the phosphotransfer activity of the fusion protein G-pp56Ick with (i) similar amounts of bacterial pp56lck obtained upon cleavage of the GST fragment with protease (as in ref. 20) and (ii) similar amounts of G-pp561ck immunopurified by using anti-pp56lCk antibodies (as in ref. 21). The specific kinase activity present in the various preparations was comparable (data not shown). Unfortunately, protease treatment caused a significant amount of a pp56ick breakdown product, and immunopurification failed to purify pp56ck to homogeneity. Purification of the wild-type and mutant G-pp561ck proteins using glutathione beads yielded =50 ng of pure G-pp561ck from 100 pug of total soluble bacterial protein. To determine the enzymatic parameters of G-pp561ck, the concentration of purified enzyme was estimated by SDS/ PAGE followed by Coomassie blue staining. Fifty nanograms of G-pp561ck was mixed with various amounts of ATP and enolase and subjected to kinase reaction. A time course of the reaction revealed that the incorporation of phosphate was linear at least for the first 5 min (data not shown). Therefore, for all the assays, 2-min incubations were used to remain in the linear range. To measure the Km of G-pp561ck for ATP, enolase concentration was fixed at 5.5 puM and ATP concentration was varied from 0.25 to 10 ,M (corresponding to 5 x Km of G-pp561ck for ATP). To calculate the Km for enolase, ATP concentration was fixed at 5 A&M and enolase was varied from 0.34 to 22 ,uM (corresponding to 3 Km of G-pp56Ick for enolase). To evaluate the phosphotransfer activity, we determined the apparent Vn for enolase phosphorylation in the presence of excess ATP (5 ,uM ATP, corresponding to Sx K.n). Enzyme and substrate were resolved by SDS/PAGE. To estimate initial velocity, X
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the amount of phosphate incorporated into enolase was measured by liquid scintillation counting. The data were evaluated by Eisenthal-Cornish-Bowden (24), and Lineweaver-Burk (25) approximations, which yielded similar values in every case. The values of apparent Km and V,: obtained for bacterial G-pp561ck using enolase as a substrate were as follows: Km ATP = 0.97 + 0.20 ,uM (mean ± SD), Km enolase = 5.58 ± 0.87 AuM, and Vma,, = 22.74 + 1.56 nmol/(min-mg) (Table 1). The G-pp561ck preparation had an apparent Km for ATP similar to that of purified baculoviral pp56Ick (D. Winkler, personal communication). With regard to the apparent Vma different values have been reported even for the same enzyme preparation, depending on the substrate analyzed (highest values have been obtained with T-cell receptor {-chain peptides; ref. 27). However, when compared under the same reaction conditions, baculoviral pp56lck and bacterial G-pp561ck displayed similar phosphotransfer activities
Analysis of pp563& Mutants Containing Single Amino Acid Substitutions in Subdomain H. To determine whether the decreased kinase activity of the single amino acid substitution mutants in residues Lys-269 to Lys-276 (Fig. 1) was due to a decrease in the binding ofATP or, alternatively, to a decrease in the efficiency of the phosphotransfer reaction, we evaluated the kinetic parameters of the mutants in vitro. Mutants were purified (as above) and the Km for ATP and Vm,, for enolase were determined (as above). In agreement with previous studies performed with pp6Osrc and epidermal growth factor receptor (11, 12, 28), mutations at the position 273 of pp5sck rendered the kinase inactive. Substitutions in all of the other positions, between 269 and 276, yielded partially active proteins. The Km for ATP of each of the mutants was similar to the Km for ATP of wild-type pp5s6ck (Table 1). In contrast, every substitution yielded a pps6Ick protein with lower phosphotransfer activity than wild-type G-pp561ck. The similarity of the Km for ATP of conservative and nonconservative substitution mutants within subdomain II suggests that this area is not likely to be responsible for ATP binding. In addition, the fact that the enzyme phosphotransfer efficiency is significantly altered when residues in the vicinity of Lys-273 are substituted suggests that Lys-273/ subdomain II is involved in the process of phosphate transfer. Comparison of the ATP-Binding Ability of Wild-Type G-pp56'C and Lys-273 -+ Arg Substitution Mutant. The kinetic analysis of the single-substitution mutants of subdomain II suggested that this subdomain is not directly involved in anchoring ATP. If this is the case, the inactive mutant at position 273 should be able to bind ATP with similar efficiency compared with wild-type pp56lck. To determine whether this hypothesis was correct, we chose to use ATP Table 1. Analysis of the kinetics of subdomain II mutants of G-pp56Ick KmK., ,utMV AM Y~~~~max. Substitution* ATP Enolase nmol/(min-mg) Wild type 0.97 ± 0.20 5.58 ± 0.87 22.74 ± 1.56 K273Xt ND ND 0 V272A (-1) 1.92 ± 0.36 1.86 ± 0.28 0.50 ± 0.02 S274N (+1) 2.06 ± 0.53 5.61 ± 0.75 2.90 ± 0.16 A271S (-2) 0.95 ± 0.35 10.12 ± 3.58 5.68 ± 4.56 L275M (+2) 0.83 ± 0.25 4.77 ± 1.59 3.66 ± 0.76 V270L (-3) 1.61 ± 0.25 7.74 ± 0.71 18.95 ± 2.54 K276V (+3) 1.08 ± 0.59 9.00 ± 2.29 6.46 ± 0.74 K269N (-4) 1.28 ± 0.42 2.92 + 0.58 6.24 + 0.72 *See Fig. 1A. tX = R (Arg), M (Met), or Asn (N).
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Proc. Natl. Acad. Sci. USA 90 (1993)
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of purified wild-type or Lys-273 -* Arg mutant G-pp561ck were analyzed, the mutant at position 273 bound an amount of 8-azido-ATP similar to the amount bound by the wild-type enzyme (Fig. 4B). In contrast, GST incubated under similar conditions yielded a small background signal (Fig. 4B). The small signal obtained when wild-type G-pp561ck was incubated in the absence of UV light (Fig. 4B) corresponds to the residual phosphotransfer activity of pp56lck at the temperature of incubation (40C). The signals of 8-azido-[32P]ATP incorporated into wild-type and mutated pp56Ick were due to specific ATP binding, as judged by the decrease in these signals observed upon addition of EDTA, absence of Mn2 , or addition of excess nonradioactive ATP (data not shown). Upon subtraction of the background signal, the ratio of mutant to wild-type signal (cpm/cpm) was calculated from five different experiments. The value obtained, 0.98 + 0.33, indicates that a similar amount of ATP reacts with wild-type enzyme and with the Lys-273 -* Arg mutant.
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DISCUSSION The results indicate that single amino acid substitutions in the area of Lys-273 affect the ability of pp56lck to transfer phosphate to a protein substrate but do not significantly alter its ability to bind ATP. Two motifs have been classically implicated in the regulation of the ATP binding: the glycine-rich loop (10), and the conserved lysine of subdomain II (Lys-72 of cAMP kinase, Lys-273 of pp56lck; refs. 8 and 9). Initially these sites were defined by labeling of the cAMP-dependent kinase with acetic anhydride: Lys-47 (next to the Gly-Xaa-Gly-Xaa-XaaGly motif) and Lys-72 were protected by MgATP against modification with acetic anhydride (10). These elegant studies provided information about which areas of a kinase were proximal of ATP, but did not delineate the specific role these areas. Mutagenesis revealed that both of each to the were for the kinase to be active (10, 11, 28, regions 29), but againrequired did not establish specific roles. To study the functional role of the conserved lysine of subdomain II (Lys-273 of pp56ock), we chose to prepare random mutations in each of the residues located between Lys-269 and Lys-276 of pp561ck. The central interest of our analysis was to distinguish between a passive role in ATP binding and an active role for Lys-273 in the phosphotransfer reaction. However, this was not the only residue mutated, since previous reports have demonstrated that mutations in this residue inactivate the kinase (11, 12, 28), making enzymatic determinations impossible. To evaluate the affinities of the mutants for ATP and their kinase activities, we measured the apparent Km for ATP and Vmax for enolase phosphorywe compared the ability of wild-type lation. In the inactive mutant Ml (Lys-273 - Arg) to bind pp56lck andaddition, an ATP analog (8-azido-ATP, an ATP analog with the crosslinking group at carbon 8 of the adenosine). From these studies, we learned that single amino acid substitutions in the microenvironment of Lys-273 induced a decrease in the kinase activity without significantly altering the affinity for ATP. In addition, the inactive Ml mutant (Lys-273 -+ Arg) bound 8-azido ATP as efficiently as wild-type pp56lck, indicating that the conserved lysine of subdomain II was not essential for binding of ATP. In agreement with our observations, substitution of Ala for Lys-116 of the yeast cAMPdependent kinase (corresponding to Lys-72 of the bovine enzyme) generated an enzyme with only residual phosphotransfer activity (103 times lower kcat) but with a similar affinity for ATP (4-fold higher Km; ref. 30). The kinase reaction includes three steps: (i) binding ofATP and protein substrate, (ii) delivery of phosphate from the ATP molecule to the protein substrate, and (iii) release of reaction products. The decreased kinase activity (Vma,) ofthe
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Biochemistry: Carrera et al.
substitution mutants in the microenvironment of Lys-273 might be caused by a direct inhibition of the phosphate transfer or, alternatively, by a decreased ability to release the products (ADP and phosphorylated protein substrate). However, since the conserved lysine seems to interact primarily with the phosphate that is transferred during the kinase reaction (see below), we favor the hypothesis that the altered phosphotransfer activity of these mutants is due to a direct inhibition of the phosphate transfer. This inhibition could be due to the alteration of Lys-273 positioning or, alternatively, to a direct role in catalysis for residues surrounding Lys-273. The latter possibility is very unlikely, since the strongest inhibitory effect was found when residues adjacent to Lys273 were substituted. These residues are expected to be buried within the protein core (6), and internal residues generally do not participate in catalysis. The altered transference of the y phosphate by the mutants might be caused by a direct effect on the interaction between Lys-72 and the y-phosphate or, alternatively, be the consequence of an altered interaction of the kinase with the a and f phosphates, which in turn would affect the y-phosphate positioning. One interpretation of our data is that the glycine-rich loop is the motif primarily responsible for ATP anchoring. That the Kd of the cAMP-dependent kinase for adenosine is only 3- to 4-fold greater than its Kd for ATP (9) suggests that the adenosine, anchored by the glycine-rich motif, is the principal area of the ATP involved in the binding to the kinase. The function of the conserved lysine of subdomain II might then be to orientate appropriately the y-phosphate and/or facilitate its transfer. When the side chain of this residue (substitution of arginine for lysine) or its physical location in the globular protein (mutations in the adjacent residues) is altered, the mutated enzyme still binds ATP with a similar affinity (Table 1 and Fig. 4), but its ability to transfer phosphate is impaired (Table 1 and Figs. 2 and 4). Our results can be rationalized by considering previous data. First, while the glycine-rich loop is found to interact with nontransferable phosphates in the nucleotide (14), Lys-72 of cAMP kinase seems to be located closer to the phosphate that is transferred (y phosphate; refs. 6, 7, and 9). Second, several lines of evidence suggest that Lys-72 contacts may differ somewhat in the presence and absence of ATP, as would be expected for a residue involved in catalysis. Studies performed with dicyclohexyl carbodiimide (31) indicate that in the absence of ATP, Lys-72 seems to interact primarily with Asp-184. In addition, the structure of the PKA catalytic domain confirms that, in the absence of ATP, these residues are localized in close proximity (6). In contrast, in the presence of ATP, Lys-72 seems to interact primarily with the -y-phosphate since (i) Lys-72 reacts with the crosslinking group of FSBA (located at a position similar to the y-phosphate; ref. 8); (ii) analysis of the cocrystal of the cAMP catalytic domain with a peptide and ATP has localized the ATP molecule in a cleft formed between the C-terminal lobe (containing Asp-184) and the N-terminal lobe (containing Lys-72), and in this complex, Asp-184 and Lys-72 are found to be close to the y-phosphate (6, 7); and (iii) studies with Beuzoadenosine 5'-triphosphate have confirmed the proximity of Lys-72 and the -phosphate in the presence of ATP (9). In summary, the observations presented here demonstrate that the conserved lysine of subdomain II is not directly involved in anchoring ATP. Our results also indicate that alterations in the positioning or side chain of this conserved lysine diminish the kinase activity of the enzyme, suggesting that this residue may have an active role in the mechanism of phosphate transfer.
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